Methods and compositions

Abstract

The invention provides various approaches to improve assisted reproductive technologies, specifically to reduce premature sister chromatid separation and aneuploidy. The approaches uses various engineered proteins that either comprise sacrificial separase sites, recruit phosphatase to cohesin, or comprise various interaction domains to result in a physical tether between different chromosomes.

Description

[0001] METHODS AND COMPOSITIONS

[0002] Field

[0003] The invention is in the field of assisted reproduction technologies.

[0004] Background

[0005] Treatments under the umbrella of assisted reproductive technologies (ART), such as in vitro fertilisation (IVF), encompass medical approaches allowing couples with infertility to conceive children. While ART enables many infertile couples to become pregnant, effective options for treating age-related female infertility are currently lacking. Whether trying to conceive naturally or when using ART, eggs are less capable of supporting healthy embryo development as women become older. In their 20’s, on average around 80% of women’s eggs have the potential to support a pregnancy. By the mid-30’s, more than half of women’s eggs are no longer capable of supporting a healthy pregnancy, and by the 40’s most, if not all, eggs are infertile. While timing may vary between individuals, the eggs of all women experience a decline in quality with age that underlies the concurrent onset of age-related female infertility.

[0006] Eggs from younger women normally contribute only one copy of each 23 human chromosomes to the embryo. By contrast, embryos generated from older eggs frequently inherit an incorrect number of chromosomes. The gain or loss of chromosomes contributed to the embryo creates a condition known as aneuploidy, leading to embryos usually failing to develop into a healthy pregnancy. Aneuploidy causes most cases of infertility and miscarriage, and underlies congenital conditions such as Down syndrome. Protecting against aneuploidy may restore fertility to older eggs and promote healthy pregnancies.

[0007] Eggs mature from long-lived cells termed oocytes. Oocytes are formed prior to birth and remain dormant within the ovary for up to decades of life. Around the time of ovulation, oocytes are reactivated to undergo a specialised cell division to mature into fertilizable eggs (first meiotic division). Following fertilisation by a sperm, a second cell division is triggered in the egg (second meiotic division), and the concurrent formation of the embryo. These first and second meiotic cell divisions are crucial for reducing the chromosome number of the oocyte by half to produce a haploid gamete. Oocytes can be decades-old at the time of fertilisation, and errors during the cell divisions of meiosis I and II often result in aneuploidy. In older eggs, aneuploidy is primarily caused by a chromosome error termed the premature separation of sister chromatids (PSSC). This error greatly affects shorter chromosomes including (but not limited to) human chromosomes 15, 16, 21 and 22 and is attributable to the gradual loss of cohesin complexes that keep chromosome structures intact. Cohesins are protein complexes responsible for tethering chromosomes together by entrapping two strands of chromosomal DNA. During cell division, they enable accurate chromosome segregation by resisting the tensile forces of the meiotic spindle until chromosomes are ready to divide. Reproductive ageing leads to the gradual and irreversible displacement of cohesin complexes from chromosomes. At a critical threshold, low cohesin levels result in chromosome structures that prematurely deteriorate into their chromatid subunits and can no longer be accurately coordinated by the meiotic spindle. Thus, prematurely separated sister chromatids risk being transmitted to the embryo randomly during meiotic cell divisions, potentiating the incidence of aneuploidy. Smaller chromosomes already have low cohesin levels due to their short lengths and are most affected by cohesin loss during aging. Indeed, trisomy of chromosome 21 (the second shortest chromosome) results in children born with Down syndrome. Meanwhile, aneuploidies involving other chromosomes usually prevent the embryo from developing further, blocking implantation or resulting in miscarriage, and effectively contributing to infertility.

[0008] Chromosome segregation error types arising in the first meiotic and second meiotic divisions of human oocyte maturation are well known and the resulting inheritance outcome of each scenario are well known. Furthermore, aneuploidy rates in human eggs vary by age and the proportion of aneuploidy meiosis-II human oocytes in different female age groups are shown in Figure IB of Gruhn et al., 2019, PMID: 31604276.

[0009] Medical treatments such as IVF cannot currently assist in preventing aneuploidy and approximately 80% of IVF failures are attributable to aneuploidy. World-wide the majority, if not a large portion, of IVF patients are women aged 35 years and above. Their eggs are at high risk of chromosome errors that generate embryos with aneuploidy. These patients seek assisted reproductive healthcare, but are generally the least likely to benefit.

[0010] Summary of the invention

[0011] The inventors have devised compositions and methods of reducing the risk of early embryonic aneuploidy and improving successful outcomes of IVF. Using microinjection- based methods common to the embryology lab, a solution containing purified artificial protein is injected into mature germinal vesicle (GV) stage and early metaphase I (MI) stage oocytes. The oocytes are matured in vitro to metaphase II stage eggs, and can be fertilised according to standard procedures.

[0012] There are several mechanisms by which the present inventors have identified the risk of aneuploidy can be mitigated, or mitigated to some extent. These mechanisms include reducing the cleavage of cohesin complexes by separase using various means; and increasing the physical association between sister chromatids, i.e. mimicking the effect of native cohesin and / or protecting endogenous cohesin. Each of these approaches may be used alone, or in combination.

[0013] Detailed description of the invention

[0014] Separase, cohesin complexes, and shugoshin-protein phosphatase 2A complexes are proteins / protein complexes critically involved in the regulation of chromosome segregation during cell division, ensuring that each daughter cell receives the correct number of chromosomes. Exemplary illustrations of these different approaches are shown in Figure 1-4 and are not to be taken as limiting.

[0015] Cohesin

[0016] Cohesins are protein complexes functioning to hold sister chromatids together following DNA replication. Cohesins are essential for accurate chromosome alignment and segregation during meiotic cell divisions. In human eggs, cohesin complexes consist of at least three core proteins that form a ring-like structure encircling the two strands of sister chromatid DNA. These proteins include two structural maintenance of chromosome (SMC) proteins (e.g. SMC1B and SMC3) and one kleisin subunit protein (e.g. Rec8). Together, cohesin complexes effectively tether sister chromatids to form a single chromosome.

[0017] In germ cells, homologous recombination allows for the exchange of alleles between homologous chromosomes. Homologous pairs of sister chromatids align by the synaptonemal complex pathway and exchange genomic DNA, forming crossover bridges between them. At completion, four chromatids are tethered together by cohesin complexes, all in one forming a structure unique to germ cells termed the bivalent chromosome.

[0018] The bivalent is a chromosome configuration essential for the accurate segregation of chromosomes in meiosis I and meiosis II. Its structure ensures that (i) homologous chromatids are oriented correctly towards opposing poles of the meiotic spindle, and; (ii) sister chromatids orient together to the same spindle pole. The bivalent chromosome also contains defined cohesin populations that ensure the orderly separation of chromosomes during first and second meiotic divisions. Accessory proteins promote or inhibit cohesin cleavage by separase at specific times of the two meiotic divisions. Cohesin regulation varies depending on the location of cohesins on the bivalent chromosome. Three separated domains have been identified thus far: centromeric, pericentromeric, and arm / distal cohesin. How cohesin complexes in each chromosome domain are regulated for timed resolution (i.e. separase cleavage) is described in further detail below.

[0019] Oocytes resume meiosis in response to hormonal signals in preparation to be ovulated and possibly fertilised. Among the first events is that the oocyte nucleus (germinal vesicle; GV) breaks down to release condensed chromosomes into the cytoplasm in a process termed nuclear envelope breakdown (NEBD). During metaphase I, chromosomes are aligned by the meiotic spindle apparatus and the spindle migrates to the cell periphery. Within 12 to 24 hours of NEBD in human eggs, anaphase of meiosis I (anaphase I) is triggered. Here, homologous chromosomes are segregated apart: the meiotic spindle pulls on each of the opposing homologous chromosomes, and; the proteolytic enzyme separase becomes catalytically active, cleaving the cohesin subunit protein, Rec8. Both centromeric and arm / distal cohesins are cleaved by separase while cohesins holding sister chromatids remain intact. Homologous chromatids are liberated from their cohesin tethers and are pulled apart to either side of the meiotic spindle. One set of homologous chromosomes is expelled into the first polar body, while the other set is retained by the oocyte. To keep sister chromatids intact, pericentromeric cohesin is normally resistant to separase cleavage and remain intact up until the second meiotic division. Pericentromeric cohesins are protected by the Shugoshin-PP2A complex that dephosphorylates Rec8 and prevents it from cleavage by separase.

[0020] After completion of anaphase I, pericentromeric cohesins keep sister chromatids intact, ensuring correct chromosome alignment by the second meiotic spindle apparatus in meiosis II. Chromosomes align to a second meiotic spindle, where fertilisation by a sperm triggers anaphase II, and a second round of spindle tension and separase activation. Shugoshin-PP2A no-longer protects cohesins, leading to cleavage Rec8 in the remaining cohesins by separase. The two sister chromatids are separated into the second polar body or retained by the newly formed zygote. The mis-regulation of cohesin cleavage- especially pericentromeric cohesin- risks disrupting the ordered division of chromosomes during meiosis I and II.

[0021] Separase

[0022] Separase is a protease enzyme that becomes active in the intervals of anaphase I and II. One of its major functions is to cleave the Rec8 subunit of cohesin, but also cleaves a variety of other substrates. Separase effectively releases homologous chromosomes or sister chromatids from cohesin tethers, allowing for coordinated division by the meiotic spindle apparatus to opposing cells (egg & first polar body; zygote & second polar body).

[0023] The activation of separase is tightly regulated. It is kept inactive by an inhibitory protein called securin that binds to the separase active site and surrounding regions. At the prescribed time, triggered by the anaphase-promoting complex (APC / C), securin is ubiquitinated at its N-terminus and degraded, freeing separase from inhibition to perform its proteolytic functions. The regulated activation of separase ensures that chromosome separation occurs only when all chromosomes are correctly aligned and attached to the spindle, preventing aneuploidy, and ensuring accurate chromosome segregation. Separase further undergoes autocatalytic cleavage releasing it from inhibition by factors such as securin.

[0024] Phosphorylation of Rec8 primes it for cleavage by separase. The cohesin subunit Rec8 can be phosphorylated by several kinases, including Polo-like kinases (Plk), casein kinase 16 / e (CK1), and Dbf4-dependent Cdc7 kinase (DDK). Phosphorylation makes Rec8 susceptible to binding and cleavage by separase. This process is crucial during anaphase I and II of meiosis, where cohesins must be cleaved to allow homologous chromosomes (anaphase I) or sister chromatids (anaphase II) to separate.

[0025] Shuaoshin-PP2A

[0026] Throughout meiosis, phosphorylation regulation coordinates when and where cohesin complexes are cleaved. During anaphase I, cohesins are protected at pericentromeric regions while triggered for removal from centromeric regions closest to kinetochores and at chromosome arm regions (arm / distal cohesin). Shugoshin (Sgol; Sgo2; generally, Sgo) proteins bind to phosphorylated threonine 120 and / or serine 121 of histone 2A located at pericentromeric regions. Sgos recruit phosphatase PP2A complexes, which dephosphorylate cohesin at peri-centromeric regions, protecting Rec8 from separase cleavage during meiosis I. The protection of pericentromeric cohesins ensures that sister chromatids remain together until meiosis II, at which point Sgo protection disappears. Another mechanism of cohesin regulation involves Meikin, a protein that binds CenpC in chromosome kinetochores and centromeric regions, functioning to recruit Polo-like kinase 1 (Plkl). Meikin is thought to promote cohesin phosphorylation and cleavage in Sgo-protected regions. Meikin is also cleaved by separase in anaphase I and understood to be essential for alignment of chromosomes in meiosis II. Overall, the precise localisation and activity of cohesin phospho- regulatory complexes dictates where and when separase cleaves cohesin Rec8.

[0027] In mammalian oocytes, Shugoshin 2 is primarily responsible for recruitment of Protein Phosphatase 2A (PP2A). PP2A is a serine / threonine phosphatase containing a scaffold A subunit, regulatory B subunit, and catalytic C subunit. PP2A including the B' variant is enriched at chromosomes pericentromeric regions of mammalian germ cell chromosomes. The c-terminal alpha-helices of two Sgo proteins can interact with the PP2A B' and C subunits. The holoenzyme complex interacts with Sgo proteins, meaning A, B, and C subunits likely assemble together prior to binding Sgo. PP2A is then recruited to the appropriate pericentromeric regions based on Sgo localization, and histone 2A phosphorylation pattern. While Sgo interacts with the PP2A B and C subunits, other proteins recruit PP2A using different interaction domains. For example, Striatin-3, part of the STRIPAK complex, recruits PP2A by binding the PP2A A subunit.

[0028] The interactions between cohesins, separase, and Sgo-PP2A is a finely tuned process that ensures the faithful segregation of chromosomes during meiotic cell division. Cohesin holds the chromosomes together until the cell is ready to divide. Separase activation ensures that the chromatids or homologous chromosomes are released at the correct time, and Shugoshins protect cleavage of pericentromeric cohesins in anaphase I. Events in anaphase I are especially critical. For example, low levels of cohesin complexes coupled with activated separase and spindle tension forces leads chromosomes to prematurely separate into chromatids, generating PSSC errors, and aneuploidy. Errors in the regulation or function of either cohesin or separase can also promote the premature separation of chromosomes, also resulting in a risk of the embryo acquiring aneuploidy.

[0029] The inventors have devised means of using or modulating or artificially mimicking these natural processes to reduce incidence of premature sister chromatid segregation and aneuploidy. Any of the various means, methods and approaches described herein may be used in isolation, or may be combined with each other to form a combination therapy or combination approach.

[0030] Anaphase Safeguard

[0031] In a first approach, the inventors have found that it is possible to decrease the frequency of segregation errors and / or increase the number of error-free oocytes by using an artificial or non-naturally occurring or exogenous engineered protein that binds to the DNA and / or chromatin and is cleaved by, and / or bound by, separase. The understanding is that this reduces cleavage of the remaining cohesin that is present in an oocyte and that is holding the chromosomes together. In this way, the number of legitimate, naturally present cohesin molecules that are cleaved by separase is reduced since the active site of separase is occupied with sacrificial cleavage sites and / or docking sites. The artificial or non-naturally occurring or exogenous engineered separase substrate protein comprises one domain that binds to the DNA or chromatin, and a second domain that comprises at least one separase cleavage substrate (i.e. at least one separase cleavage site and / or at least one separase docking site). Upon introduction of the artificial or non-naturally occurring or exogenous engineered separase substrate protein (also known as Anaphase Safeguard) the artificial or non- naturally occurring or exogenous engineered separase substrate protein binds to the chromatids, coating them with the artificial or non-naturally occurring or exogenous engineered separase cleavage sites and / or separase docking sites.

[0032] By "separase cleavage substrate", we include a substrate that is cleavable by separase and / or can be bound by separase. Preferably the substrate is cleavable by separase and / or can be bound by separase during anaphase. A separase cleavage substrate may comprise at least one separase cleavage site (also referred to herein as separase cleavage motif) and / or at least one separase docking site (also referred to herein as separase docking motif). In some embodiments, the separase cleavage substrate does not comprise a separase inhibitory motif.

[0033] For separase to cut cohesin subunits (like Rec8 or Sccl), it recognises specific cleavage sites in its substrates. These cleavage motifs often contain an ExxR consensus, but separase also requires additional docking motifs to bind substrates efficiently. In certain embodiments, the engineered polypeptide comprises an LPE motif (Leu-Pro- Glu), which serves as a conserved docking element or pseudo-binding site or docking site for separase, allowing separase to cleave the conserved nearby cleavage site. The presence of the LPE motif facilitates binding of separase to the protein, thereby enhancing recognition and subsequent proteolytic cleavage at one or more separase cleavage sites located within the protein. In some embodiments the LPE motif may increase the efficiency and / or specificity of cleavage by separase. The LPE motif is therefore not a cleavage site perse, but its presence is considered to be advantageous in sequestering separase to the engineered protein and away from native sites.

[0034] There are many separase cleavage motifs. Separase cleavage motifs tend to have a motif of ExxR where x is any amino acid. Exemplary separase cleavage motifs are SEQ ID NO: 126 ETVEEERA, SEQ ID NO: 127 EIEVLREA, SEQ ID NO: 128 SPELFR, SEQ ID NO: 137 LVPEDLRK, SEQ ID NO: 138 ENPEVPRE, SEQ ID NO: 139, EPIIEEPSRL, SEQ ID NO: 140 DSVMEASRT, SEQ ID NO: 141 LEIPRLP, SEQ ID NO: 142 AEEERRK, SEQ ID NO: 147 GPEIMRSI, SEQ ID NO: 148 SFEILRGS, SEQ ID NO: 149 ECEVLRRD, SEQ ID NO: 151 AEEPKRRG, SEQ ID NO: 152 RHCEERRPQ, SEQ ID NO: 153 EWELLRLD, SEQ ID NO: 155 ALETLRRV, SEQ ID NO: 157 EDELYRQS, SEQ ID NO: 159 DVEENRTE, SEQ ID NO: 160 ETEAERE, SEQ ID NO: 162 VVNELFRDG, SEQ ID NO: 164 RRAEQRRTK, SEQ ID NO: 165 DYRESERA, SEQ ID NO: 166 ETQESPRCS, SEQ ID NO: 167 SFEFERV, SEQ ID NO 168: TQERKRE, SEQ ID NO: 169 REEKRK, SEQ ID NO: 170 EKENSRRI, SEQ ID NO: 171 RKETFRKV, SEQ ID NO: 172 IHENDRGS, SEQ ID NO: 173 GKEGSRPA, SEQ ID NO: 174 NTEMQRNK, SEQ ID NO: 175 SSESAREP, SEQ ID NO: 176 SSEKKRER.

[0035] Accordingly, the invention provides an engineered protein comprising a first domain that is a nucleic acid binding domain and at least a second domain that is a separase cleavage substrate domain comprising at least one amino acid sequence a) that is cleavable by a separase; and / or b) that can be bound by separase.

[0036] The key requirement of this approach is that amino acid sequences that are cleavage sites for separase and / or docking sites for separase are brought into proximity with the nucleic acid, for example proximity to the chromosomes. This may be achieved in various ways, for example the engineered protein could bind directly to the DNA, for example to the chromatin, or could bind indirectly to the DNA for example chromatin via an interaction with an endogenous DNA binding protein. Accordingly, in some instances the nucleic acid binding domain is a domain that is capable of binding to an endogenous nucleic acid binding protein, for example to an endogenous chromatin binding protein. Preferably the first domain is a nucleic acid binding domain that is capable of directly binding to nucleic acid, for example directly to the chromatin or DNA. The first domain that is a nucleic acid binding domain may be a domain that binds directly to nucleic acid, for example may bind directly to chromatin or DNA. The skilled person will understand that the terms "chromatin" and "DNA" may be employed interchangeably, unless otherwise specified. While chromatin refers to the higher-order nucleoprotein complex of DNA associated with histones and other structural or regulatory proteins, DNA generally refers to the nucleic acid polymer itself. Similarly, chromatin-binding domains (CBDs), such as the C-terminal chromatin binding domain of mouse Ki67, are protein domains that typically recognize and associate with DNA in the context of nucleosomes and chromatin structure, whereas DNA-binding domains generally refer to protein domains that recognize and bind DNA independently of chromatin context. For the purposes of this disclosure, however, references to chromatin-binding domains and DNA-binding domains are used interchangeably, and both are intended to encompass domains that facilitate association with DNA, whether in nucleosomal, chromatin, or naked DNA form. Preferably however the nucleic acid binding domain is a domain that binds to chromatin.

[0037] The arrangement of the first domain that is a nucleic acid binding domain, or domain binding to an endogenous nucleic acid binding protein, and at least a second domain that is a separase cleavage substrate domain, comprising of at least one amino acid sequence cleavable by a separase, may be in any arrangement. For example, the nucleic acid binding domain may be N-terminal or C-terminal to the separase cleavage substrate domain.

[0038] The nucleic acid binding domain can be any domain that can bind to a nucleic acid, or any domain binding to an endogenous nucleic acid binding protein. In the instance of designing the engineered protein for the purposes of reducing premature sister chromatin separation, the nucleic acid binding domain should be a DNA binding domain, for example that binds to chromatin. Accordingly, preferably the nucleic acid binding domain is a DNA binding domain and preferably binds to chromatin.

[0039] The DNA binding domain, for example chromatin binding domain, may be any domain capable of directly binding to DNA such as chromatin, or any domain binding to an endogenous nucleic acid binding protein. In some instances the DNA binding domain is a domain that is capable of binding along the length of a chromosome, for example capable of binding to the chromatin along the length of the chromosome. In other instances the DNA binding domain is a domain that preferentially binds to specific regions, such as the centromeric or pericentromeric regions. In some instances the nucleic acid binding domain capable of directly binding to nucleic acid is selected from the group comprising or consisting of: a) a leucine-arginine rich domain, optionally Ki67 DNA-binding domain, optionally SEQ ID NO: 1; b) an alpha-solenoid domain, optionally Transcription activator-like [TAL] effector domain optionally SEQ ID NO: 2; c) a basic helix-turn-helix; d) a zinc finger; e) a leucine zipper; f) a winged helix; g) a winged helix-turn-helix; h) helix-loop-helix; i) HMG-box; j) Wor3 domain; and / or k) a histone protein.

[0040] In some instances the domain capable of binding to a nucleic acid binding protein is: a) a chromo-domain, for example a Chromobox protein, preferably the human Chromobox protein 5 (CBX5; Heterochromatin Protein 1 alpha) SEQ ID NO: 3; or b) a phosphorylated Histone binding domain.

[0041] An example of a DNA binding domain that binds along the length of the chromosome is the Ki67 DNA binding domain. An example of a DNA binding domain that binds to centromeric and to pericentromeric regions of a chromosome is the TAL effector. An example of a domain binding to a nucleic acid binding protein is the CBX5 protein.

[0042] In some embodiments the nucleic acid binding domain capable of directly binding to nucleic acid is selected from the group comprising or consisting of: a) the c-terminal domain of Ki-67, optionally the terminal 326 (amino acids 2930 - 3256) of human Ki-67, optionally has an amino acid sequence of SEQ ID NO.l, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.l; or b) the DNA binding alpha-solenoid domain from the TAL01 engineered protein, optionally has an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.2; or c) an amino acid sequence of SEQ ID NO.2, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.2.

[0043] In some instances the domain capable of binding to a nucleic acid binding protein is: a) a methylated histone binding domain from human Chromobox protein 5 (CBX5) optionally has an amino acid sequence of SEQ ID NO: 3, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.3; or b) a phosphorylated Histone 2A binding domain from human Shugoshin 2 optionally has an amino acid sequence of SEQ ID NO: 4, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.4.

[0044] The skilled person will appreciate that where a specific sequence is given, it is well within the skill of the skilled person to produce mutants or other variants of the sequence that retain the required function. For example it is appreciated that a protein sequence with a level of variation to the claimed sequence will still accomplish the aims of the invention. This applies to this particular approach, but also applies to reference to any sequence described herein, for example to the approaches set out under Anaphase Shield, Turbo-Linker and Mono-Linker below. Accordingly, for any sequence described herein, reference to a sequence also includes a sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the specified sequence.

[0045] The skilled person will appreciate that for this approach to work, the engineered protein must comprise one or more amino acid sequences that is recognisable and capable of being bound by the separase that is naturally present in the target cell. For example where the target cell is a human oocyte, the amino acid sequence must be recognisable and capable of being bound by the separase protein present in a human oocyte. Similarly, where the target cell for instance a camel oocyte, then the amino acid sequence must be recognisable and capable of being bound by the separase protein present in a camel oocyte.

[0046] In some instances the amino acid sequence is capable of being recognised, bound and cleaved by the separase. In other instances the amino acid sequence is capable of being recognised and bound, but not cleaved by the separase. Such an embodiment is still considered to be useful since the separase enzyme present in the target cell will still be occupied by these additional sites, whether they are cleaved or not.

[0047] In some embodiments the engineered protein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs. For example in some embodiments the second domain that is a separase cleavage substrate domain comprising at least one amino acid sequence a) that is cleavable by a separase; and / or b) that can be bound by separase comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs. The at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs are, in preferred embodiments, positioned appropriately between 50 to 125 amino acids C-terminally relative to the separase cleavage site, for example positioned relative to a ExxR motif, such that separase binds to the engineered protein and in some instances cleaves the cleavage site. In some embodiments the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs are present anywhere in the engineered protein. Preferably the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs are located in the second domain that is a separase cleavage substrate domain comprising at least one amino acid sequence a) that is cleavable by a separase; and / or b) that can be bound by separase. Accordingly any reference throughout to the second domain that is a separase cleavage substrate domain comprising at least one amino acid sequence a) that is cleavable by a separase; and / or b) that can be bound by separase is intended to encompass a domain that in some embodiments comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs.

[0048] The at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs may, in preferred embodiments, be arranged in tandem. For example the engineered protein for example the second domain may comprise a sequence of LPELPE [SEQ ID NO: 124] or LPELPELPE [SEQ ID NO: 125].

[0049] In some embodiments the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs are not arranged directly in tandem, for example are not arranged in an LPELPELPE fashion but instead have a spacer sequence between them, for example have a spacer nucleic acid sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues.

[0050] In some embodiments the engineered protein, for example the second domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs and also comprises one or more separase cleavage motifs for example with an ExxR consensus for example may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs and also comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more separase cleavage motifs for example with an ExxR consensus. In some embodiments the engineered protein, for example the second domain of the engineered protein, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs and does not comprise one or more separase cleavage motifs for example with an ExxR consensus.

[0051] In some embodiments the engineered protein, for example the second domain of the engineered protein, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more separase cleavage motifs for example with an ExxR consensus, and does not comprise one or more LPE motifs.

[0052] In some embodiments the preferred engineered protein contains a sequence of SEQ ID NO: 7, SEQ ID NO: 40, SEQ ID NO: 51, SEQ ID NO: 59.

[0053] In some preferred embodiments the engineered protein, for example the second domain of the engineered protein, comprises two separase cleavage motifs and a tandem LPE motif of sequence LPELPE. In some embodiments the two separase cleavage motifs have the same sequence. In other embodiments the two separase cleavage motifs have different sequences. In some embodiments a first separase cleavage motif has a sequence of SEQ ID NO: 126 and a second separase cleavage motif has a sequence of SEQ ID NO: 127. Accordingly, in some embodiments the engineered protein, for example the second domain of the engineered proteins comprises a sequence of SEQ ID NO: 126, a sequence of SEQ ID NO: 127, and a sequence of SEQ ID NO: 124 (LPELPE).

[0054] To be cleaved by separase, the Rec8 domain must be phosphorylated. The inventors have surprisingly found that it is possible to mimic phosphorylation of the Rec8 domain by substituting one or more residues with negatively charged amino acids. Accordingly in some embodiments the invention provides a Rec8 domain that comprises one or more substitutions to a negatively charged amino acid, for example to a aspartic acid. The engineered Rec8 domain may be from any species. Preferably the engineered Rec8 domain is from a human or a mouse. In preferred embodiments the Rec8 domain is a region of Rec8 that comprises both a LPELPE sequence, and two separase cleavage motifs.

[0055] In some embodiments the engineered Rec8 domain has a sequence that comprises or consists of SEQ ID NO: 51 or 7 but where one or more residues has been substituted to a negative amino acid, for example to an aspartic acid, relative to the sequence of SEQ ID NO: 51 or 7, or comprises or consists of a sequence with at least 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or 100% sequence identify to SEQ ID NO: 51 or 7 but where one or more residues has been substituted to a negative amino acid, for example to an aspartic acid, relative to the sequence of SEQ ID NO: 51 or 7. In some embodiments one or more serine and / or threonine residues of SEQ ID NO: 51 or 7 are substituted with a negative amino acid, for example aspartic acid.

[0056] In some embodiments the engineered Rec8 domain has a sequence that comprises or consists of SEQ ID NO: 59 or 120 or comprises or consists of a sequence with at least 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or 100% sequence identify to SEQ ID NO: 59 but residues that are an aspartic acid in SEQ ID NO: 59 are retained as an aspartic acid.

[0057] In some embodiments where the engineered Rec8 domain has a sequence identity that is less than 100% to a specified sequence it will be appreciated that the engineered Rec8 domain should retain the ability to be bound by separase and / or cleaved by separase. In the present context it is not considered that any other function of Rec8 is required.

[0058] Accordingly in some embodiments the second domain comprises or consists of a sequence of SEQ ID NO: 59 or 120.

[0059] In some embodiments the second domain comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more negatively charged residues. In some embodiments some or all of the negatively charged residues are aspartic acids.

[0060] The amino acid sequence that is recognised and bound by separase, or that is cleavable by separase, may be a naturally occurring sequence, or may be an artificial or engineered sequence, for example may be a rationally designed sequence.

[0061] In some instances, the amino acid sequence that is recognised and bound by separase, or that is cleavable by separase, is selected from the group comprising or consisting of: a) an amino acid sequence cleavable by human separase; b) the separase binding site from human Securin of SEQ ID NO.5 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.5; c) the separase cleavage site from human Meikin of SEQ ID NO.6 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.6; d) the separase cleavage site from human Rec8 of SEQ ID NO.7 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.7; and / or e) the separase cleavage site derived from human Rec8 containing substitutions of threonines and / or serines for bulky amino acids, preferably aspartate, mimicking phosphorylated threonines and / or serines of SEQ ID NO.8 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.8; f) an amino acid sequence cleavable by separase belonging to human, murine, other rodent, equine, bovine, porcine, ovine, canine, feline, marsupial, camelid, nonhuman primate, northern white rhinoceros, southern white rhinoceros, African elephant, Asian elephant, endangered or threatened mammalian animal species; g) an engineered Rec8 domain that has a sequence that comprises or consists of SEQ ID NO: 59 or 120 or comprises or consists of a sequence with at least 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or 100% sequence identify to SEQ ID NO: 59 but residues that are an aspartic acid in SEQ ID NO: 59 are retained as an aspartic acid; and / or h) an engineered Rec8 domain that comprises or consists of a sequence of SEQ ID NO: 59 or 120.

[0062] In some embodiments the engineered protein comprises a single amino acid sequence that is capable of being recognised and bound by, or cleaved by, a separase.

[0063] However it is also considered to be useful if the engineered protein comprises multiple amino acid sequences that are capable of being recognised and bound by, or cleaved by, a separase. In this way, a single engineered protein molecule, once introduced into the oocyte, may occupy the active site of a number of separase molecules, enhancing the intended effect.

[0064] Accordingly in some embodiments, the artificial separase substrate protein domain comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid sequences capable of being recognised and bound by, or cleaved by, a separase. In some instances at least two of the amino acid sequences that are capable of being recognised and bound by, or cleaved by, a separase have different amino acid sequences. For example, the engineered protein may comprise two different amino acid sequences capable of being recognised and bound, or cleaved, by two different separases. Such an embodiment may also comprise multiple copies of one or more sequences that are the same.

[0065] In other embodiments all of the amino acid sequences capable of being recognised and bound by, or cleaved by, a separase are the same amino acid sequence.

[0066] In some embodiments, the engineered protein comprises one reporter domain. A reporter domain may be a fluorescent moiety, e.g. GFP RFP, YFP, mCherry, mNeon Green, mScarlet or EGFP. A reporter domain may also be an epitope tag, e.g. FLAG, 6xHis, Streptavadin.

[0067] It should be clear to the skilled person that the engineered protein is intended to be a therapeutic protein, or to have an advantageous effect on IVF for instance. The engineered protein is not simply a sensor protein. For example in some embodiments the engineered protein does not comprise one or more reporter domains. For example, in some embodiments the engineered protein does not comprise two or more domains that are reporter domains. In some embodiments the engineered protein does not comprise one or more reporter domains that is a fluorescent protein domain. In some embodiment the engineered protein does not comprise two reporter domains, wherein either or both reporter domains are fluorescent protein domains. In some embodiments the engineered protein does not comprise a domain that is any one or more of: GFP RFP, YFP, mCherry, mNeon Green, mScarlet or EGFP.

[0068] Preferably, the DNA or chromatin binding protein is not one that disrupts the chromatin structure.

[0069] In some embodiments, the DNA binding domain in the engineered protein is not or does not comprise H2B and / or CENP-B (e.g. as the DNA binding domain). In some embodiments, the engineered agent does not comprise H2B and / or CENP-B (e.g. as the DNA binding domain) and one or more reporter domains.

[0070] H2B exhibits toxicity at high concentrations and is therefore not considered to be suitable for use in therapeutics. In some embodiments, the engineered protein does not comprise:

[0071] H2B and / or CENP-B (e.g. as the DNA binding domain);

[0072] - one or more reporter domains; and

[0073] Meikin, Rec8, Rad21 and / or Sccl or a polypeptide fragment thereof comprising at least one separase cleavage site (e.g. as a separase cleavage substrate).

[0074] In some embodiments, the polypeptide fragment of Meikin, Rec8, Rad21 and / or Sccl consists of:

[0075] - residues 1-332 of Meikin;

[0076] - residues 115-235 of Meikin

[0077] - residues 376-548 of Rad21;

[0078] - residues 297-506 of Rec8;

[0079] - residues 142-467 of Sccl; or

[0080] - residues 107-268 of Sccl.

[0081] In some embodiments, the engineered protein does not comprise one or more of the following :

[0082] (i) H2B or a fragment thereof that retains the DNA binding ability; one or more reporter domains; and Meiken or a fragment thereof comprising one or more separase cleavage sites (e.g. as a separase cleavage substrate),

[0083] (ii) H2B or a fragment thereof that retains the DNA binding ability; one or more reporter domains; and Rad21 or a fragment thereof comprising one or more separase cleavage sites (e.g. as a separase cleavage substrate),

[0084] (iii) H2B or a fragment thereof that retains the DNA binding ability; one or more reporter domains; and Rec8 or a fragment thereof comprising one or more separase cleavage sites (e.g. as a separase cleavage substrate),

[0085] (iv) H2B or a fragment thereof that retains the DNA binding ability; one or more reporter domains; and Sccl or a fragment thereof comprising one or more separase cleavage sites (e.g. as a separase cleavage substrate),

[0086] (v) CENP-B or a fragment thereof that retains the DNA binding ability; one or more reporter domains; and Meiken or a fragment thereof comprising one or more separase cleavage sites (e.g. as a separase cleavage substrate),

[0087] (vi) CENP-B or a fragment thereof that retains the DNA binding ability; one or more reporter domains; and Rad21 or a fragment thereof comprising one or more separase cleavage sites (e.g. as a separase cleavage substrate), (vii) CENP-B or a fragment thereof that retains the DNA binding ability; one or more reporter domains; and Rec8 or a fragment thereof comprising one or more separase cleavage sites (e.g. as a separase cleavage substrate),

[0088] (viii) CENP-B or a fragment thereof that retains the DNA binding ability; one or more reporter domains; and Sccl or a fragment thereof comprising one or more separase cleavage sites (e.g. as a separase cleavage substrate),

[0089] (ix) H2B-mScarlet-hMeikin-mNeonGreen,

[0090] (x) H2B-mScarlet-hRad21-mNeonGreen,

[0091] (xi) H2B-mScarlet-hRec8-mNeonGreen,

[0092] (xii) H2B-mScarlet-hMeikin (residues 1-332, optionally with S149A mutation)- mNeonGreen,

[0093] (xiii) H2B-mScarlet-hRad21 (residues 142-476)-mNeonGreen,

[0094] (xiv) H2B-mScarlet-hRec8 (residues 297-506)-mNeonGreen,

[0095] (xv) H2B-mCherry-Sccl-EGFP,

[0096] (xvi) CENP-B-mCherry-Sccl-EGFP,

[0097] (xvii) H2B-mCherry-Sccl (residues 142-467)-EGFP,

[0098] (xviii) CENP-B-mCherry- Sccl (residues 142-467)-EGFP,

[0099] (xix) H2B-mCherry-Sccl-YFP, and

[0100] (xx) H2B-mCherry-Sccl (residues 107-268 aa)-YFP.

[0101] In some embodiments, the separase cleavage site is ExxR.

[0102] Once the engineered protein has been introduced into an oocyte, for example via microinjection, the engineered protein protects the cohesin present on the chromosomes. There are various consequences of this that can be measured, so as to allow the skilled person to determine whether the desired effect has been achieved.

[0103] For example, in some embodiments when the engineered protein is present in oocytes: a) the proportion of chromosome premature separated sister chromatids (PSSC) decreases, optionally significantly decreases; b) wherein the amount of cohesin does not decrease, or does not significantly decrease, measurable by quantitative or semi-quantitative laboratory methods; c) the proportion of chromosome non-disjunction increases, optionally significantly increases in eggs matured from treated oocytes compared to untreated oocytes; d) the proportion of chromosome non-disjunction increases, optionally significantly increases in oocytes from females genetically predisposed for subfertility or infertility; and / or e) the proportion of chromosome non-disjunction increases or significantly increases, in oocytes modified by pharmacological substances or introduction of proteins causing the oocytes to be predisposed to chromosome instability; f) the proportion of error-free oocytes or embryos is increased.

[0104] Specifically, by "error-free oocytes" we include the meaning of oocytes in which the oocytes comprise the correct allocation of chromosomes, where all chromatid pairs are correctly associated (not dissociated).

[0105] In some embodiments administration of the engineered protein(s) of the invention results in a decrease in the frequency of dissociation of sister chromatid pairs, which can present as sister chromatids with kinetochores that are located 4 pm or more from each other, or when kinetochores of sister chromatids are aligned perpendicular to the spindle axis, or whereby the number of whole chromosome aneuploidies are increased, as measured by sequencing of first and / or second polar bodies, and / or oocyte and first and / or second polar body trios, or by sequencing of preimplantation embryos using techniques such as preimplantation genetic testing for aneuploidy (PGT-A).

[0106] By "present in oocytes" we typically include the meaning of "upon introduction of", or "upon administration of" since in typical embodiments the engineered proteins will be produced and subsequently administered to the oocyte, for example via microinjection, electroporation, optical poration, or vesicular fusion, i.e. the protein itself if administered into the oocyte. However there are some instances where "present in oocytes" may encompass "expressed in" the oocyte. For example in some instances the engineered protein may be administered to the oocyte in an RIMA form, in which case the engineered protein will be generated within the oocyte. Similarly there may be instances where it is the DNA that encodes the engineered protein that is introduced transiently into the oocyte, either as a plasmid, or non-replicating artificial chromosome. Alternatively, but less preferably, there may be instances where it is the DNA that encodes the engineered protein that is introduced into the oocyte either episomally or via genomic integration.

[0107] In some preferred embodiments the invention does not provide for the genomic integration of nucleic acid encoding the engineered protein of the invention into the genome of an oocyte.

[0108] Accordingly in some embodiments, when the engineered protein is administered to an oocyte, for example via microinjection: a) the proportion of chromosome premature separated sister chromatids (PSSC) decreases, optionally significantly decreases; b) wherein the amount of cohesin does not decrease, or does not significantly decrease, measurable by quantitative or semi-quantitative laboratory methods; c) the proportion of chromosome non-disjunction increases, optionally significantly increases in eggs matured from treated oocytes compared to untreated oocytes; d) the proportion of chromosome non-disjunction increases, optionally significantly increases in oocytes from females genetically predisposed for subfertility or infertility; e) the proportion of chromosome non-disjunction increases or significantly increases, in oocytes modified by pharmacological substances or introduction of proteins causing the oocytes to be predisposed to chromosome instability; and / or f) the proportion of error-free oocytes is increased.

[0109] These are all parameters that are readily testable by the skilled person. The skilled person will also appreciate that the above references to an increase or a decrease are an increase or decrease relative to an appropriate control, for example relative to an oocyte in which the engineered protein is not present.

[0110] All combinations of nucleic acid binding domains, for example DNA binding domain, and domains binding endogenous nucleic acid binding proteins, for example DNA binding proteins, and amino acid sequence that is able to be recognised and bound by, or cleaved by, a separase, are contemplated by the present invention. For instance, the engineered protein may comprise a DNA binding domain that comprises or consists of the Ki67 DNA-binding domain, for example SEQ ID NO: 1, or the Transcription activator-like [TAL] effector domain for example of SEQ ID NO: 2, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.l or 2; and tThe amino acid sequence recognised and bound by, or cleavable by a separase may be the separase cleavage site from human Meikin, for example of sequence SEQ ID NO.6 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.6.

[0111] The skilled person will appreciate that other features may be present in the engineered protein. For example the engineered protein may comprise linkers, as is standard when creating fusion proteins. The engineered protein may also comprise one or more protein tags, for example that are useful in protein purification, or for visualisation using fluorescence microscopy.

[0112] Anaphase Shield

[0113] Another separate or complementary means to protect the existing cohesin present on the chromatids involves modulating the phosphorylation status of cohesin. The phosphorylation of cohesin plays a critical regulatory role in its cleavage by separase during cell division, affecting its stability and the timing of its cleavage, which is essential for accurate chromosome segregation.

[0114] The inventors have found that stimulating the recruitment of phosphatase activity to the chromatids protects cohesin from cleavage by separase. The recruitment of the phosphatase activity may be a PP2A activity, or may be another phosphatase activity. The recruitment may be either via the introduction to an oocyte of a fusion protein that comprises both DNA binding activity and phosphatase activity, e.g. a fusion of a DNA binding domain to PP2A or a subunit of PP2A; or by the introduction of a protein that comprises a DNA binding domain fused to the alpha-helical Sgo-PP2A interaction peptide, which would then subsequently recruit the PP2A activity to the chromatids; or by the introduction of a protein that comprises a domain binding to an endogenous DNA binding protein fused to the alpha-helical Sgo-PP2A interaction peptide.

[0115] Accordingly the invention provides an engineered protein comprising at least a first domain that is i) a nucleic acid binding domain capable of directly binding to nucleic acid or ii) a domain capable of binding to a nucleic acid binding protein; and at least a second domain that comprises at least one amino acid sequence that recruits at least one phosphatase activity.

[0116] Preferences for the first domain that is i) a nucleic acid binding domain capable of directly binding to nucleic acid or ii) a domain capable of binding to a nucleic acid binding protein are as described elsewhere herein. For example, preferably the nucleic acid binding domain is a DNA binding domain, or domain binding to an endogenous DNA binding protein.

[0117] As set out elsewhere, the first domain may be a DNA binding domain, for example that can bind directly to DNA, or may be a domain that is capable of binding to an endogenous DNA binding protein. In some instances the DNA binding domain is a domain that preferentially binds to specific regions of a chromosome, such as the centromeric and / or pericentromeric regions. In some instances the DNA binding domain is a domain that is capable of binding along the length of a chromosome.

[0118] In some instances the nucleic acid binding domain capable of directly binding to nucleic acid is selected from the group comprising or consisting of: a) a leucine-arginine rich domain, optionally Ki67 DNA-binding domain, optionally SEQ ID NO: 1; b) an alpha-solenoid domain, optionally Transcription activator-like [TAL] effector domain optionally SEQ ID NO: 2; c) a basic helix-turn-helix; d) a zinc finger; e) a leucine zipper; f) a winged helix; g) a winged helix-turn-helix; h) helix-loop-helix; i) HMG-box; j) Wor3 domain; and / or k) a histone protein.

[0119] In some instances the domain capable of binding to nucleic acid binding protein is: a) a chromo-domain, for example a Chromobox protein, preferably the human Chromobox protein 5 (CBX5; Heterochromatin Protein 1 alpha) SEQ ID NO: 3; or b) a phosphorylated Histone binding domain.

[0120] An example of a DNA binding domain that binds along the length of the chromosome is the Ki67 DNA binding domain. An example of a DNA binding domain that binds to centromeric and to pericentromeric regions of a chromosome is the TAL effector. An example of a domain binding to a nucleic acid binding protein is the CBX5 protein.

[0121] In some embodiments the nucleic acid binding domain capable of directly binding to nucleic acid is selected from the group comprising or consisting of: a) the c-terminal domain of Ki-67, optionally the terminal 326 (amino acids 2930 - 3256) of human Ki-67, optionally has an amino acid sequence of SEQ ID NO.l, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.l; or b) the DNA binding alpha-solenoid domain from the TAL01 engineered protein, optionally has an amino acid sequence of SEQ ID NO.2, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.2; or c) an amino acid sequence of SEQ ID NO.2, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.2.

[0122] In some instances the domain capable of binding to a nucleic acid binding protein is: a) a methylated histone binding domain from human Chromobox protein 5 (CBX5) optionally has an amino acid sequence of SEQ ID NO: 3, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.3; or b)a phosphorylated Histone 2A binding domain from human Shugoshin 2 optionally has an amino acid sequence of SEQ ID NO: 4, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.4.

[0123] Since the focus is to protect cohesin, and since cohesin is located primarily at the centromeric and pericentromeric regions, in preferred embodiments the DNA binding domain is a domain that is able to bind to the centromeric and pericentromeric regions, or preferably binds to these regions.

[0124] In some instances, the second domain comprises just one amino acid sequence that recruits a phosphatase activity.

[0125] In some embodiments the second domain comprises at least two or more amino acid sequences that recruit at least one phosphatase activity. The second domain may comprise any number of amino acid sequences that recruit at least one phosphatase activity, for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more such sequences.

[0126] At least two or more amino acid sequences that recruit at least one phosphatase activity may be different amino acid sequences and recruit different phosphatases; or may be the same amino acid sequences and recruit multiple molecules of the same phosphatase.

[0127] The one or more amino acid sequences that recruit at least one phosphatase activity may be any suitable sequence, for example may be a portion of a protein that interacts with phosphatase proteins. In some embodiments the amino acid sequence that recruits a phosphatase activity may comprise or consist of any one or more of: a) the human Shugoshin 1 PP2A interaction peptide of SEQ ID NO.10 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.10; b) the human Shugoshin 2 PP2A interaction peptide of SEQ ID NO.11 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.11; c) the human Striatin 3 PP2A interaction peptide of SEQ ID NO.12 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.12; d) the human PP2A scaffold subunit A (PPP2A alpha) protein of SEQ ID NO.13 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.13; e) the human PP2A regulatory subunit B' (PPP2R5C) protein of SEQ ID NO.14 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.14; and / or, f) the human PP2A catalytic subunit C (PPP2C alpha) protein of SEQ ID NO.15 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.15; g) the mouse Shugoshin-2 Coiled-Coil Protein Sequence [SEQ ID NO: 130] or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 130; h) the Mouse Striatin-3 Coiled-Coil Protein Sequence [SEQ ID NO: 131] or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 131; or i) Mouse Shugoshin-1 Coiled-Coil Protein Sequence [SEQ ID NO: 132] or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 132.

[0128] In some embodiments the amino acid sequence that recruits a phosphatase activity may comprise tandem repeats of a sequence, for example of a sequence of any of SEQ ID NO: 10-15 or 130-132, or of an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the specified sequence. In some embodiments the tandem repeats are separated by a linker sequence, for example a linker of sequence GGGGS (G4S).

[0129] In some embodiments the amino acid sequence that recruits a phosphatase activity comprises three tandem repeats of a sequence of any of SEQ ID NO: 10-15 or ISO- 132 each separated by a linker of GGGG, or of an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the specified sequence.

[0130] In some embodiments the amino acid sequence that recruits a phosphatase activity comprises or consists of a sequence of any of SEQ ID NO: 74, 78, 82, 107, 111 or 113, or of an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the specified sequence.

[0131] In some embodiments, the engineered protein is for use in a host cell and so should be able to recruit a phosphatase activity in said host cells.

[0132] Accordingly in some preferred embodiments, when the engineered protein is present in a host cell, the second domain recruits a phosphatase activity. For example, when the host cell is a mammalian cell, for example a human cell, a murine cell; a rodent cell; an equine cell; a bovine cell; a porcine cell; an ovine cell; a canine cell; a feline cell; a marsupial cell; a camelid cell; a cell of a non-human primate; a cell of a northern white rhinoceros; a cell of a southern white rhinoceros' a cell of an African elephant; a cell of an Asian elephant; a cell of an endangered or threatened mammalian animal species - the engineered protein when present in these cells recruits a phosphatase activity.

[0133] The engineered protein must also be capable of binding to DNA and recruiting a phosphatase activity simultaneously, since the aim is to bring the phosphatase activity into close proximity with cohesin to protect it from degradation.

[0134] The skilled person can readily test the ability of an engineered protein of the invention to both bind to DNA, and to recruit a phosphatase activity. For example, by visualising the engineered protein and to detect phosphorylation levels by immunocytohistochemistry methods in conjunction with fluorescence microscopy. The phosphatase activity may be any phosphatase activity capable of dephosphorylating the rec8 subunit of cohesin. For example in some preferred embodiments the recruited phosphatase is a PP2A complex phosphatase activity.

[0135] As for the Anaphase Safeguard embodiment described elsewhere herein, the presence of the engineered protein protects cohesin and reduces premature sister chromatid segregation and aneuploidy. Accordingly, when the engineered protein is present in an oocyte: a) the proportion of phosphorylated cohesin is decreased, optionally significantly decreased, measurable by determining a decrease in phosphorylated endogenous proteins in regions located at or near to the engineered protein, and / or a decrease in chromosome premature separated sister chromatids (PSSC); b) the amount of cohesin does not decrease, optionally does not significantly decrease, measurable by quantitative or semi-quantitative laboratory methods; c) the proportion of chromosome non-disjunction increases, optionally significantly increases, in eggs matured from treated oocytes compared to untreated oocytes; d) the proportion of chromosome non-disjunction increases, optionally significantly increases, in oocytes from females genetically predisposed for subfertility or infertility; and / or e) the proportion of chromosome non-disjunction increases, optionally significantly increases in oocytes modified by pharmacological substances or introduction of proteins causing the oocytes to be predisposed to chromosome instability.

[0136] As for Anaphase Safeguard, by "present in oocytes" we typically include the meaning of "upon introduction of", or "upon administration of" since in typical embodiments the engineered proteins will be produced and subsequently administered to the oocyte, for example via microinjection, electroporation, or vesicular fusion. However there are some instances where "present in oocytes" may encompass "expressed in" the oocyte. For example in some instances the engineered protein may be administered to the oocyte in an RIMA form, in which case the engineered protein will be generated within the oocyte. Similarly there may be instances where it is the DNA that encodes the engineered protein that is introduced transiently into the oocyte, either as a plasmid, or non-replicating artificial chromosome.

[0137] The skilled person will appreciate that the introduction of artificial separase substrate sites (e.g. as described elsewhere herein under Anaphase Safeguard), and the recruitment of phosphatase activity to the DNA (as described here for Anaphase Shield) are not mutually exclusive. In some instances, in use, two separate proteins that each fulfill one of these modes of action may be administered (e.g. injected into an oocyte or expressed within the oocyte) to an oocyte. However, in some instances a single protein may comprise both properties.

[0138] Accordingly the invention also provides an engineered protein that comprises a nucleic acid binding domain (preferably a DNA binding domain), a domain that is a separase cleavage substrate domain comprising at least one amino acid sequence cleavable by a separase, and a domain that comprises at least one amino acid sequence that recruits at least one phosphatase activity. Exemplary arrangements are shown in Figure 2B. Since the separase cleavage domain is intended to be cleaved by separase, preferably the separase cleavage domain is positioned between the DNA binding domain and the domain that comprises at least one amino acid sequence that recruits at least one phosphatase activity, enabling dissociation of the DNA binding domain from the domain recruiting the phosphatase activity by the completion of anaphase I, where in preferably the separase cleavage domain is modified to mimic phosphorylated serines and / or threonines of SEQ ID NO.8 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.8

[0139] Preferences for the nucleic acid binding domain, domain that is a separase cleavage substrate domain, and the domain that comprises at least one amino acid sequence that recruits at least one phosphatase activity are as described elsewhere herein.

[0140] Turbo-linker

[0141] The above approaches (set out under Anaphase Safeguard and Anaphase Shield) aim to stabilise the existing cohesin associated with the chromatids. Further approaches that can be used in isolation, or in combination with either the Anaphase Safeguard and / or Anaphase Shield approaches involves the introduction of proteins that mimic the chromatin-association function of cohesin. These approaches (set out under TurboLinker and MonoLinker) use a system that comprises DNA binding domains that bind to regions of DNA and physically links them together. This can either be by the administration of a single protein complex that comprises one DNA binding domain, or by the administration of two different proteins described here under TurboLinker (which in some instances may be administered as a single complex). The result is a polymeric, or oligomeric, network of engineered proteins and chromatin, strengthening the interactions between chromosomes. Together, DNA-binding proteins connect together by covalently interacting protein domains to physically connect chromosomal DNA together.

[0142] The invention provides an engineered protein comprising: a) a nucleic acid binding domain, preferably a DNA binding domain; b) a first interaction domain capable of interacting with a first partner domain; and c) a second interaction domain capable of interacting with a second partner domain wherein the first interaction domain and second interaction domains are not able to interact with each other within the same protein molecule.

[0143] Preferences for the nucleic acid binding domain are as set out elsewhere herein. For example preferably the nucleic acid binding domain is a DNA binding domain. The DNA binding domain may be selected from the group comprising or consisting of: a) a leucine-arginine rich domain, optionally Ki67 DNA-binding domain, optionally SEQ ID NO: 1; b) an alpha-solenoid domain, optionally Transcription activator-like [TAL] effector domain optionally SEQ ID NO: 2; c) a phosphorylated Histone binding domain; d) a basic helix-turn-helix; e) a zinc finger; f) a leucine zipper; g) a winged helix; h) a winged helix-turn-helix; i) helix-loop-helix; j) HMG-box; k) Wor3 domain.

[0144] The arrangement of the nucleic acid binding domain and the two interactions domains can be any arrangement, Figure 3A shows some exemplary arrangements. For instance in some embodiments the domains are arranged in the following order, N- terminal to C-terminal:

[0145] DNA binding domain; first interaction domain; second interaction domain DNA binding domain; second interaction domain; first interaction domain First interaction domain; second interaction domain; DNA binding domain Second interaction domain; first interaction domain; DNA binding domain First interaction domain, DNA binding domain, second interaction domain Second interaction domain, DNA binding domain, first interaction domain.

[0146] Preferably the domains are arranged in the following order, N-terminal to C-terminal:

[0147] DNA binding domain; first interaction domain; second interaction domain DNA binding domain; second interaction domain; first interaction domain First interaction domain; second interaction domain; DNA binding domain Second interaction domain; first interaction domain; DNA binding domain.

[0148] The first interaction domain and the second interaction domain have different sequences - i.e. the first interaction domain cannot be the same as the second interaction domain since the mode of action requires two or more proteins to a) bind to DNA; and b) bind to / interact with each other; and c) preferably interact with multiple other molecules of the engineered protein(s) so as to allow the formation of a polymeric, or oligomeric, network of protein and DNA.

[0149] One approach uses a single engineered protein that comprises or consists of a DNA binding domain and at least a first and a second interaction domain, where the first and second interaction domain are able to interact with one another between protein molecules, i.e. inter-protein interactions, but are not able to interact with one another within a protein molecule, i.e. are not able to generate intra-protein interactions. To be clear, the first (or second) interaction domain of a first engineered protein is able to interact with the second (or first) interaction domain of a second engineered protein; and the second (or first) interaction domain of a first engineered protein is able to interact with the first (or second) interaction domain of a second engineered protein. The skilled person will appreciate that such an arrangement allows the formation of a polymeric, or oligomeric, network, for example with the first interaction domain of a first engineered protein interacting with the second interaction domain of a second engineered protein, and for example the first interaction domain of the second engineered protein to interact with the second interaction domain of a third engineered protein. The second interaction domain of the first engineered protein may interact with the first interaction domain of a fourth engineered protein. Since each engineered protein also has a DNA binding domain, it can readily be seen how a network of proteins and chromosomes can be formed, holding the chromatids together. A key feature is that the first and second interaction domains must not be capable of intra-molecular interaction when not in the presence of the host intracellular environment i.e. the first and second interaction domains are either not capable of interacting with one another by virtue of their amino acid sequences, or are prevented from interacting with one another where the amino acid sequences of the interaction domains would otherwise allow an interaction.

[0150] This does not mean that the first and second interaction domains cannot both be from an interacting pair of domains (e.g. a SpyTag / SpyCatcher pair, or a pair of interacting coiled-coil domains). However, when the protein has been produced, e.g. in solution and prior to administration to an oocyte, where the first and second interaction domains are both part of an interacting pair, means must be taken to prevent the domains from interacting until such a point as the engineered protein is administered to an oocyte. Suitable means for preventing protein-protein interactions are described elsewhere herein. For example in some embodiments the engineered protein comprises a further domain that allows for steric effects, for example from physically preventing the first and second interaction domains to contact one another sufficiently to allow, for example, an isopeptide bond to form between the interaction domains.

[0151] Accordingly, in some embodiments, the first partner domain is the second interaction domain and the second partner domain is the first interaction domain, and wherein said first interaction domain is capable of interacting with said second interaction domain between engineered protein molecules, i.e. in some embodiments the first interaction domain and the second interaction domain are from the same interacting pair, i.e. are capable of interacting between protein molecules with one another by virtue of their amino acid sequences. In this embodiment, only a single injection of this single protein into an oocyte is considered to be required. Upon administration to an oocyte, the DNA binding domain binds to DNA and the first and second interaction domains interact with each other, between protein molecules. In some instances the proteins may interact as set out in Figure 3B, with the first and second interaction domains of a first engineered protein molecule both interacting with the corresponding interaction domains of a second engineered protein molecule. However, preferably 3, 4, 5 or more different protein molecules will all be linked together, with corresponding regions of DNA - for example the first interaction domain of a first engineered protein molecule may interact with the second interaction domain of a second engineered protein molecule, and the second interaction domain of the first engineered protein molecule may interact with the first interaction domain of a third engineered protein molecule, and so on. However, in some embodiments the first interaction domain and the second interaction domain interact with different partner domains, i.e. the first interaction domain is part of a first interacting pair, and the second interaction domain is part of a second, different interacting pair. In this embodiment a first engineered protein is deployed in combination with a second engineered protein that comprises a DNA binding domain and two different interaction domains that each interact with the interaction domains of the first protein. Although this embodiment may require the separate administration of at least two different engineered proteins, it has benefits in that the manufacture of two engineered proteins is straightforward, and there is no requirement for means to prevent protein-protein interactions prior to administration to the oocyte.

[0152] Accordingly in this embodiment the first partner domain and the second partner domain have different amino acid sequences since they are designed to partner with different interaction domains.

[0153] The interaction between a) the first and second interaction domain (in the previous embodiment where the first and second interaction domains are designed to interact with one another); b) the first interaction domain and the first partner domain; and c) the second interaction domain and the second partner domain may be any kind of interaction, for example may be a covalent or a non-covalent interaction.

[0154] However, since the aim is to tether chromosomes together, a strong interaction is preferred. Accordingly it is preferred that upon interaction between any of the domains and the corresponding partner domain, a covalent bond is formed.

[0155] In some embodiments then: the first interaction domain and the first partner domain are a pair of domains that upon interaction form a covalent bond between the two domains; the second interaction domain and the second partner domain are a pair of domains that upon interaction form a covalent bond between the two domains; and where the first partner domain is the second interaction domain and the second partner domain is the first interaction domain, and wherein said first interaction domain is capable of interacting with said second interaction domain between engineered protein molecules, the first interaction domain and the second interaction domain are a pair of domains that upon interaction form a covalent bond between the two domains.

[0156] Preferably: when the first interaction domain interacts with the first partner domain, a covalent bond is formed between the first interaction domain and the first partner domain; when the second interaction domain interacts with the second partner domain, a covalent bond is formed between the second interaction domain and the second partner domain; and where the first partner domain is the second interaction domain and the second partner domain is the first interaction domain, and wherein said first interaction domain is capable of interacting with said second interaction domain between protein molecules, when the first partner domain interacts with the second partner domain, a covalent bond is formed between the first interaction domain and the second interaction domain.

[0157] The skilled person is aware of suitable interacting pairs of domains that form covalent bonds upon interaction. For example pairs of domains that spontaneously form an isopeptide bond upon interaction, forms an ester bond upon interaction, or forms a thioester bond upon interaction.

[0158] Exemplary interacting domains are the SpyTag / SpyCatcher pair; DogTag / DogCatcher pair and SnoopTag / SnoopCatcher, and the later iterations of these domains such as SpyTag002 / SpyCatcher002 and SpyTag003 / SpyCatcher003. Reference to any of SpyTag / SpyCatcher pair; DogTag / DogCatcher pair and SnoopTag / SnoopCatcher pair are intended to also include within the meaning the later iterations and improved variants of these domains.

[0159] SpyTag and SpyCatcher were formed from the splitting and engineering of the CnaB2 domain of the FbaB protein from Streptococcus pyogenes, which naturally forms an intramolecular isopeptide bond to assist colonization of the host cell. Before the development of SpyTag / SpyCatcher, the pair Isopeptag / Pllln-C was created from protein Spy0128 of Streptococcus pyogenes. SnoopTag / SnoopCatcher was developed from the RrgA protein of Streptococcus pneumoniae and has no cross- reactivity with SpyTag / SpyCatcher. The same domain from RrgA has now been split in a different way to that used to create SnoopTag / SnoopCatcher, with the new pair called DogTag / DogCatcher. Unlike SpyTag and SnoopTag which have extended structures, the region of RrgA used to create DogTag forms a 0-halrpln and so predisposed for successful insertion into protein loops. The pair SdyTag / SdyCatcher was also developed this Tag / Catcher pair has cross-reactivity with the SpyTag / SpyCatcher. A new Tag / Catcher pair was developed from SpyTag / SpyCatcher with minimal mutations. SpyTag I3W (Aw) reacts with SpyCatcher F77V, F94A (BVA) but minimally with SpyCatcher, whereas SpyCatcher F77V, F94A can react with both SpyTag I3W and SpyTag.

[0160] A different chemistry can be exploited for protein ligation: the discovery of an intramolecular ester bond formation in Clostridium perfrlngens cell-surface adhesln protein Cpe0147 led to the development of another Tag / Catcher pair with CpeO 147565-587 as the Tag and Cpe0147439-563 as the Catcher.

[0161] Accordingly the skilled person is well aware of the development of interacting pairs of domains, and is readily able to select the most appropriate domains to use based on factors such as their cross- reactivity. Although specific exemplary domains are mentioned herein, the skilled person understands that any domain that has the required ability to form the appropriate bonds between two domains may be used, and all are encompassed herein.

[0162] In some embodiments of the engineered protein: a) the first interaction domain is selected from any of DogTag, DogCatcher, SpyTag or SpyCatcher, SnoopTag or SnoopCatcher; and / or b) the second interaction domain DogTag, DogCatcher, SpyTag or SpyCatcher, SnoopTag or SnoopCatcher.

[0163] As set out above, in one particular embodiment the first and second interaction domain are from the same interacting pair, and so in some embodiments: a) the first interaction domain is a SpyTag domain and the second interaction domain is a SpyCatcher domain; b) the first interaction domain is a DogTag domain and the second interaction domain is a DogCatcher domain; or c) the first interaction domain is a SnoopTag domain and the second interaction domain is a SnoopCatcher domain.

[0164] However, in other embodiments that are considered to have at least advantages in ease of manufacture and storage the first and second domains are not able to interact with one another based on the amino acid sequence of the domains. Accordingly in some embodiments: a) where the first interaction domain is a SpyTag domain the second interaction domain is not a SpyCatcher domain; b) where the first interaction domain is a DogTag domain the second interaction domain is not a DogCatcher domain; or c) were the first interaction domain is a SnoopTag domain the second interaction domain is not a SnoopCatcher domain.

[0165] In these embodiments then the first and second interaction domains are from different interacting pairs. For example in some embodiments the:

[0166] First interaction domain is DogTag and the second interaction domain is

[0167] SpyTag;

[0168] First interaction domain is DogTag and the second interaction domain is

[0169] SpyCatcher;

[0170] First interaction domain is DogTag and the second interaction domain is

[0171] SnoopCatcher;

[0172] First interaction domain is DogTag and the second interaction domain is

[0173] SnoopTag;

[0174] First interaction domain is DogTag and the second interaction domain is

[0175] DogTag;

[0176] First interaction domain is DogCatcher and the second interaction domain is

[0177] SpyTag;

[0178] First interaction domain is DogCatcher and the second interaction domain is

[0179] SpyCatcher;

[0180] First interaction domain is DogCatcher and the second interaction domain is

[0181] SnoopCatcher;

[0182] First interaction domain is DogCatcher and the second interaction domain is

[0183] SnoopTag;

[0184] First interaction domain is DogCatcher and the second interaction domain is

[0185] DogCatcher;

[0186] First interaction domain is SpyCatcher and the second interaction domain is

[0187] SpyCatcher;

[0188] First interaction domain is SpyCatcher and the second interaction domain is

[0189] SnoopCatcher;

[0190] First interaction domain is SpyCatcher and the second interaction domain is

[0191] SnoopTag;

[0192] First interaction domain is SpyCatcher and the second interaction domain is

[0193] DogTag;

[0194] First interaction domain is SpyCatcher and the second interaction domain is

[0195] DogCatcher;

[0196] First interaction domain is SpyTag and the second interaction domain is SpyTag; First interaction domain is SpyTag and the second interaction domain is SnoopCatcher;

[0197] First interaction domain is SpyTag and the second interaction domain is SnoopTag;

[0198] First interaction domain is SpyTag and the second interaction domain is

[0199] DogTag;

[0200] First interaction domain is SpyTag and the second interaction domain is

[0201] DogCatcher;

[0202] First interaction domain is SnoopTag and the second interaction domain is

[0203] SpyTag;

[0204] First interaction domain is SnoopTag and the second interaction domain is SpyCatcher;

[0205] First interaction domain is SnoopTag and the second interaction domain is SnoopTag;

[0206] First interaction domain is SnoopTag and the second interaction domain is

[0207] DogTag;

[0208] First interaction domain is SnoopTag and the second interaction domain is

[0209] DogCatcher;

[0210] First interaction domain is SnoopCatcher and the second interaction domain is

[0211] SpyTag;

[0212] First interaction domain is SnoopCatcher and the second interaction domain is SpyCatcher;

[0213] First interaction domain is SnoopCatcher and the second interaction domain is SnoopCatcher;

[0214] First interaction domain is SnoopCatcher and the second interaction domain is

[0215] DogTag; or,

[0216] First interaction domain is SnoopCatcher and the second interaction domain is

[0217] DogCatcher.

[0218] In some embodiments the engineered protein comprises two interaction domains. However it is also considered to be useful of the engineered protein comprises at least 3, 4, 5, 6, 7, 8, 9 or 10 or more interaction domains. Some of the interaction domains may be the same, i.e. copies of one another, whereas some of the interaction domains may be different to one another. In some embodiments the first, second, third or more interaction domains are selected from any of DogTag, DogCatcher, SpyTag or SpyCatcher, SnoopTag or SnoopCatcher. As set out above, in some instances the engineered protein comprises a first and second interaction domain that are part of the same interacting pair. This has challenges in the context of manufacturing and storage, since the interaction between the two domains must be prevented until the engineered protein is deployed into a cellular context, for example administered into an oocyte. Accordingly also provided is a composition comprising an engineered protein as described herein wherein the first and second interaction domains are prevented from interacting with one another within the composition, i.e. the composition has features or comprises further agents that prevent the interaction. It is important that the interaction is only prevented temporarily, or reversibly, since once administered to a cell such as an oocyte, the interaction domains must then be able to interact with one another so that intermolecular bonds are formed.

[0219] The invention also provides a composition comprising any one or more of the engineered proteins set out herein and which prevents interactions between the required domains. For example the invention provides a composition comprising one or more engineered proteins described herein wherein when present in the composition, the composition prevents: a) interaction between the first interaction domain and the first partner domain; b) interaction between the second interaction domain and the second partner domain; and / or c) interaction between the first interaction domain and the second interaction domain.

[0220] For example in some instances the composition may comprise two or more engineered proteins, where the interaction domain of one engineered protein is the partner domain of a second engineered protein. As for the embodiment described above, these interactions must be temporarily or reversibly prevented until such a time as the composition is employed.

[0221] There are various means of temporarily and reversibly preventing these interactions. For example the use of antibodies or aptamers to block the interaction. In a preferred embodiment the prevention of interaction is managed via pH, for example maintaining the engineered proteins in the composition at a pH that prevents the required interactions. For example the pH of the composition may be titrated to a point at which the interactions are inhibited. For example in some embodiments the composition prevents said interaction by having a low pH, for example at a pH of 5 or less, optionally 4.8, 4.6, 4.4, 4.2, 4.0, 3.8, 3.6 or less than 3.4. The invention also provides various complexes of the described engineered proteins.

[0222] For example the invention provides a protein complex comprising an engineered protein described herein wherein the first interaction domain interacts with the second interaction domain.

[0223] In other embodiments the invention provides a protein complex comprising at least a first and a second engineered protein described herein wherein the first and second engineered proteins are different and wherein at least one interaction domain of the first engineered protein interacts with at least one interaction domain of the second engineered protein. In some embodiments, e.g. prior to administration of the complex to a cell such as an oocyte, only one of the first and second interaction domains of the first engineered protein interacts with one of the first or second interaction domains of the second engineered protein.

[0224] In other embodiments the interaction domains of the engineered proteins in the complex all interact with the corresponding partner domain.

[0225] Mono-Linker

[0226] A further approach, named Mono-Linker, is provided whereby only one engineered DNA-binding protein is needed to generate a similar polymer network on chromosomes.

[0227] This approach allows for the formation of a complex prior to deployment for example into an oocyte, and allows for the administration of this single complex into a target cell, such as an oocyte, reducing the number of physical interventions to the cell (for example oocyte).

[0228] The complex is formed between at least a first engineered protein as set out under TurboLinker above, and a bridging protein.

[0229] Preferences for the engineered protein here are as set out under TurboLinker above.

[0230] Preferences for the nucleic acid binding domain (preferably DNA binding domain) are as set out elsewhere herein. Preferences for the interaction domains are set out elsewhere herein, for example under TurboLinker above. For example the interaction domains are preferably capable of forming covalent bonds such as an isopeptide bond when contacted with its corresponding partner domain.

[0231] The bridging protein comprises a first and a second partner domain. The first interaction domain of the at least first engineered protein and the first partner domain of the bridging protein have amino acid sequences that would allow an interaction between the first partner domain of the bridging protein and the first partner domain of the bridging protein; and the second interaction domain of the at least first engineered protein and the second partner domain of the bridging protein have amino acid sequences that would allow an interaction between the second partner domain of the bridging protein and the second partner domain of the bridging protein.

[0232] Accordingly the invention provides a protein complex comprising at least a first engineered protein of the invention and at least a first bridging protein, wherein said bridging protein comprises a first and a second partner domain, wherein the first interaction domain of the at least first engineered protein and the first partner domain of the bridging protein have amino acid sequences that would allow an interaction between the first interaction domain of the first engineered protein and the first partner domain of the bridging protein; and the second interaction domain of the at least first engineered protein and the second partner domain of the bridging protein have amino acid sequences that would allow an interaction between the second interaction domain of the first engineered protein and the second partner domain of the bridging protein.

[0233] In the complex: the first interaction domain of the at least first engineered protein interacts with the first partner domain of the bridging protein; but the second interaction domain of the at least first engineered protein is prevented from interacting with the second partner domain of the bridging protein.

[0234] In this way, the bridging protein is physically connected to the at least first engineered protein via one interacting pair of domains, but both the engineered protein and bridging protein has an interaction domain that has not been connected with the corresponding partner, and is free to interact with said partner once the complex is administered to a cell, for example to an oocyte, and establish a network of proteins and genomic DNA, tethering the chromatids together. In some embodiments, in the context of interactions between the engineered protein and the bridging protein, preventing the interaction using a photo-actuating protein cage domain blocking interaction such as AsLOV2 is not considered to be appropriate.

[0235] In some preferred embodiments the second interaction domain of the at least first engineered protein is embedded within the body of the first engineered protein and is not integrated to either the N or C terminal sides of the protein. Preferably, the second interaction domain is integrated within an external solvent-interacting amino acid loop of the first engineered protein, or integrated into any other part of the first engineered protein.

[0236] Accordingly in some instances the second interaction domain is not located at the N- terminus and the C-terminus of the first engineered protein. For example, by "not located at the N-terminus and the C-terminus of the first engineered protein" we include the meaning that the second interaction domain is not located within the terminal 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% or 13% of the first engineered protein.

[0237] In other embodiments by "not located at the N-terminus and the C-terminus of the first engineered protein" we include the meaning that the second interaction domain is not located within the terminal 50, 60, 70, 80, 90, 100 amino acids of the first engineered protein.

[0238] Preferably the second interaction domain is located within an external solventinteracting amino acid loop.

[0239] In some instances this embodiment is similar to an embodiment set out under TurboLinker that requires a single engineered protein that is capable of interaction between engineered protein molecules.

[0240] In some embodiments the bridging protein does not have a DNA binding domain. In this case the DNA-protein interactions are mediated solely by the nucleic acid binding domain present in the engineered protein.

[0241] As set out above there are various means of preventing the interaction between the second interaction domain and second partner domain of the bridging protein. For example the use of antibodies or aptamers to block the interaction. In a preferred embodiment the prevention of interaction is managed via pH, for example maintaining the engineered proteins in the composition at a pH that prevents the required interactions. For example the pH of the composition may be titrated to a point at which the interactions are inhibited. For example in some embodiments the composition prevents said interaction by having a low pH, for example at a pH of 5 or less, optionally 4.8, 4.6, 4.4, 4.2, 4.0, 3.8, 3.6 or less than 3.4.

[0242] For example at this low pH the DogTag and DogCatcher domains are unable to interact with one another, or only interact minimally with one another. However the SpyTag / SpyCatcher domains are able to interact at this pH, allowing the formation of a covalent bond between the engineered protein and the bridging protein, whilst leaving one interaction domain “free" for interaction upon administration to a cell such as an oocyte.

[0243] Accordingly in some embodiments the interaction between the second interaction domain of the at least first engineered protein and the second partner domain of the bridging protein is prevented by maintaining the complex at a low pH for example at a pH of 5 or less, optionally 4.8, 4.6, 4.4, 4.2, 4.0, 3.8, 3.6 or less than 3.4.

[0244] Since the prevention of the interaction should be temporary or reversible (as set out elsewhere herein), in some embodiments the at least one interaction domain of the first engineered protein is able to interact with at least one partner domain of the bridging protein at a pH of greater than 5, for example at a pH of greater than 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 or higher; but is unable to interact with at least one interaction domain of the second engineered protein at a pH of less than 5, for example at a pH of less than 4.8, 4.6, 4.4, 4.2, 4.0, 3.8, 3.6 or less than 3.4.

[0245] In other cases either the engineered protein and / or the bridging protein may be designed to as to prevent this interaction, for example the arrangement of the domains may preclude the interaction; or further domains may be introduced that physically prevent the required interaction, for example wherein the composition prevents said interaction by having a photo-actuating protein cage domain blocking interaction. For example the photo-actuating protein cage domain AsLOV2 domain is relieved from protein caging effects by exposure to blue light.

[0246] In some specific examples of this embodiment: a) the first interaction domain of the at least first engineered protein is a SpyTag domain and the second interaction domain of the at least first engineered protein is a DogTag domain; and the first partner domain is a SpyCatcher domain and the second partner domain is a DogCatcher domain; b) the first interaction domain of the at least first engineered protein is a SpyCatcher domain and the second interaction domain of the at least first engineered protein is a DogTag domain; and the first partner domain is a SpyTag domain and the second partner domain is a DogCatcher domain; c) the first interaction domain of the at least first engineered protein is a SpyTag domain and the second interaction domain of the at least first engineered protein is a DogCatcher domain; and the first partner domain is a SpyCatcher domain and the second partner domain is a DogTag domain; d) the first interaction domain of the at least first engineered protein is a SpyCatcher domain and the second interaction domain of the at least first engineered protein is a DogCatcher domain; and the first partner domain is a SpyTag domain and the second partner domain is a DogTag domain.

[0247] Also in specific examples of this embodiment the nucleic acid binding of the at least first engineered protein is a DNA binding domain and is a TAL human domain, for example the DNA binding alpha-solenoid from the TAL01 engineered protein, for example has an amino acid sequence of SEQ ID NO.2, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.2

[0248] In a more preferred embodiment: a) the nucleic acid binding of the at least first engineered protein is a DNA binding domain and is a TAL human domain, for example the DNA binding alphasolenoid from the TAL01 engineered protein, for example has an amino acid sequence of SEQ ID NO.2, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.2; and b) i) the first interaction domain of the at least first engineered protein is a SpyCatcher domain and the second interaction domain of the at least first engineered protein is a DogTag domain; and the first partner domain is a SpyTag domain and the second partner domain is a DogCatcher domain. ii) the first interaction domain of the at least first engineered protein is a SpyTag domain and the second interaction domain of the at least first engineered protein is a DogCatcher domain; and the first partner domain is a SpyCatcher domain and the second partner domain is a DogTag domain; iii) the first interaction domain of the at least first engineered protein is a SpyCatcher domain and the second interaction domain of the at least first engineered protein is a DogCatcher domain; and the first partner domain is a SpyTag domain and the second partner domain is a DogTag domain; or iv) the first interaction domain of the at least first engineered protein is a SpyTag domain and the second interaction domain of the at least first engineered protein is a DogTag domain; and the first partner domain is a SpyCatcher domain and the second partner domain is a DogCatcher domain; preferably i) the first interaction domain of the at least first engineered protein is a SpyCatcher domain and the second interaction domain of the at least first engineered protein is a DogTag domain; and the first partner domain is a SpyTag domain and the second partner domain is a DogCatcher domain.

[0249] The invention also provides a bridging protein as set out herein. The invention also provides compositions and pharmaceutical compositions comprising the bridging protein described herein.

[0250] The invention also provides a complex comprising any one or more of the engineered proteins described herein bound to nucleic acid, for example DNA, for example genomic DNA.

[0251] Such a complex may be generated in vitro, for example during testing of the engineered proteins. The complex of the invention may also be generated in an isolated oocyte, for example in an oocyte obtained from a subject. Compositions

[0252] The skilled person will appreciate that any of the engineered proteins of the invention, including those set out under Anaphase Safeguard, Anaphase Shield, Turbo-Linker and Mono-Linker can be provided as one or more compositions that comprises one or more different engineered proteins, in any combination. For example, the composition may comprise one or more engineered proteins set out under Turbo-Linker; or may comprise one or more engineered proteins set out under Turbo-Linker and one or more proteins set out under Anaphase Safeguard, i.e. an engineered protein that comprises an amino acid sequence that can be recognised and bound by, or cleaved by, a separase.

[0253] The skilled person will appreciate that none of the approaches are mutually exclusive and so provision of various engineered proteins that act by different modes of action is appropriate.

[0254] The skilled person will also realise that since the engineered proteins are typically for administration into a cell, for example into an oocyte, the compositions for administration should be pharmaceutical compositions, and comprise one or more pharmaceutical excipients or the like that make the composition suitable for use in a biological system.

[0255] Methods of producing the engineered proteins and complexes

[0256] The invention also provides various methods of producing the engineered proteins, complexes and compositions of the invention.

[0257] In particular instances for the production of a complex where one interacting pair is required to interact and a second interacting pair is prevented from interacting, the method involves providing one of the component proteins at a low pH and contacting said component protein with the second component protein, so that the one interacting pair interacts, preferably forming a covalent bond, but where the second interacting pair is prevented from interacting due to the low pH.

[0258] Accordingly the invention provides a method of producing a protein complex that comprises at least a first and a second engineered protein of the invention, wherein the first and second engineered proteins are different and wherein at least one interaction domain of the first engineered protein interacts with at least one interaction domain of the second engineered protein, optionally wherein only one of the first and second interaction domains of the first engineered protein interacts with one of the first or second interaction domains of the second engineered protein said method comprising the steps of a) providing the first engineered protein at a pH which allows interaction of one of the interaction domains of the first engineered protein with one of the interaction domains of the second engineered protein, but prevents interaction of the second interaction domain of the first engineered protein with an interaction domain of the second engineered protein, optionally wherein the first engineered protein is provided at a low pH, optionally at a pH of 5 or less, optionally 4.8, 4.6, 4.4, 4.2, 4.0, 3.8, 3.6 or less than 3.4; b) contacting the first engineered protein with the second engineered protein.

[0259] The invention also provides a method of producing the complex comprising at least a first engineered protein of the invention and at least a first bridging protein, wherein said bridging protein comprises a first and a second partner domain, Wherein the first interaction domain of the at least first engineered protein and the first partner domain of the bridging protein have amino acid sequences that would allow an interaction between the first partner domain of the bridging protein and the first partner domain of the bridging protein; and the second interaction domain of the at least first engineered protein and the second partner domain of the bridging protein have amino acid sequences that would allow an interaction between the second partner domain of the bridging protein and the second partner domain of the bridging protein and wherein in the complex: the first interaction domain of the at least first engineered protein interacts with the first partner domain of the bridging protein; but the second interaction domain of the at least first engineered protein is prevented from interacting with the second partner domain of the bridging protein, said method comprising a) providing a first engineered protein of the invention under conditions that would allow the first interaction domain of the at least first engineered protein to interact with the first partner domain of the bridging protein, but which would prevent the interaction between the second interaction domain of the at least first engineered protein and the second partner domain of the bridging protein; b) contacting said first engineered protein with the bridging protein, maintaining said conditions that allow the first interaction domain of the at least first engineered protein to interact with the first partner domain of the bridging protein, but which prevent the interaction between the second interaction domain of the at least first engineered protein and the second partner domain of the bridging protein.

[0260] As set out elsewhere, said conditions that prevent an interaction may be a low pH, optionally at a pH of 5 or less, optionally 4.8, 4.6, 4.4, 4.2, 4.0, 3.8, 3.6 or less than 3.4.

[0261] Methods of using the engineered proteins / complexes / compositions

[0262] It will be apparent to the skilled person how the various engineered proteins, complexes and compositions can be deployed to aid in assisted reproduction. In some instances these engineered proteins, complexes, and compositions may be considered to be therapeutic proteins, complexes, and compositions since they are able to treat or reduce infertility. In some instances, infertility may not be considered to be something in need of therapy, in which case the engineered proteins, compositions, and complexes are non-therapeutic engineered proteins, compositions and complexes.

[0263] It will be appreciated that instead of injecting the engineered protein itself into an oocyte, the therapeutic uses may involve the microinjection of a polynucleotide, such as i) an RIMA molecule such as mRNA that encodes the engineered protein, or ii) DNA, for example double stranded DNA or single stranded DNA that encodes the engineered protein.

[0264] Therefore the invention provides an RNA molecule, for example an mRNA molecule, that encodes any of the engineered proteins of the invention as set out herein.

[0265] The invention also provides a DNA molecule, for example a double stranded DNA molecule or single stranded DNA molecule, that encodes any of the engineered proteins of the invention as set out herein. The DNA molecule that encodes the engineered protein may be part of a larger DNA molecule such as a vector, for example a plasmid. The invention therefore also provides a DNA vector, for example a plasmid, that encodes one or more of the engineered proteins of the invention.

[0266] The invention also provides a viral vector that comprise a polynucleotide, such as an RNA or a DNA, that encodes one or more engineered proteins of the invention.

[0267] Accordingly the invention provides methods of treating or reducing infertility, wherein said method comprises administration of any one or more engineered proteins, complexes or compositions, polynucleotides, vectors or viral vectors of the invention into an oocyte, for example administered by microinjection.

[0268] In some preferred embodiments it is the protein itself that is administered to the oocyte, for example via microinjection.

[0269] The oocyte may be any oocyte. The oocyte may be a mammalian oocyte such as a human oocyte, or may be an oocyte belonging to murine, rodent, equine, bovine, porcine, ovine, canine, feline, marsupial, camelid, non-human primate, northern white rhinoceros, southern white rhinoceros, African elephant, Asian elephant, endangered or threatened mammalian animal species. The oocyte may be a frozen oocyte, for example an oocyte taken from a female at a relative young age, or taken from a female ahead of treatment that is expected to impact fertility. The oocyte may be taken from a human female that is over the age of 25, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or over 50 years of age, such as a human female that is over the age of 25, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or over 50 years of age. The oocyte may be taken from a female mouse that is between 2 to 4 months of age or 15 to 20 months of age (as per the Examples). Preferably, the oocyte is taken from a female mouse that is 16 months and above on retrieval. The human oocyte presented here was retrieved from a female patient 33 years of age. Preferably of 35 years and above where the oocyte is taken from a human female. The oocyte may be taken from a non-human female but of equivalent reproductive age to a human female over the age of 25, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or over 50 years of age, as determined by counting the equivalent number of predicted menstrual cycles, or estrous cycles, and / or ovulations of oocytes and / or eggs of the non-human female. For example, to approximate correspondence of reproductive age between human and mouse species the following calculation can be performed: while human females undergo one full menstrual cycle on average every 28 days, a female mouse undergoes one full estrous cycle approximately every 4 to 5 days. Therefore, a human female aged 30 years of age has ovulated a single oocyte approximately 200 times, while a female mouse aged 14 months has ovulated multiple oocytes approximately 100 times. Here, both human and mouse may be considered approximately similar in reproductive age. Similar calculation can be used to estimate reproductive ageing in females of different mammalian species.

[0270] Preferences for the oocyte specified here apply to references to an oocyte throughout this disclosure. The oocyte may be an oocyte equivalent in age, using the aforementioned method, to an oocyte from a human female 30, 31, 32, 33, 34, or over 35 years of age; or, may be an oocyte taken at an age where oocytes are known to exhibit errors in chromosome structure including premature separation of sister chromatids (PSSC), non-disjunction, and / or reverse segregation during maturation into eggs and / or embryos at frequencies similar to eggs or embryos matured from human females 30, 31, 32, 33, 34, or over 35 years of age; or, may be an oocyte taken at an age where a female individual or group of individuals are determined to experience sub-fertility or infertility, and optionally where the male-factor is judged not to be the cause of sub-fertility or infertility by, for example, standard semen analysis methods.

[0271] Once injected, preferably the oocyte is allowed to develop throughout fertilization and beyond.

[0272] The invention also provides any one or more engineered proteins, complexes or compositions of the invention for use in a method of treating or reducing infertility, wherein said method comprises administration of into an oocyte, for example administered by microinjection.

[0273] The invention also provides the use of any one or more engineered proteins, complexes or compositions of the invention in a method of manufacture for a medicament to treat or reduce infertility.

[0274] The invention also provides a method of reducing premature separation of sister chromatids in an oocyte, or reducing aneuploidy, said method comprising administering any one or more of the engineered proteins, compositions, or complexes of any of the invention to the oocyte. The administration may be via microinjection into the oocyte, optionally in vitro microinjection, optionally similar to the technique of intra-cytoplasmic sperm injection (ICSI) regularly used for controlled fertilisation and / or treating male-factor infertility. The method may comprise a single microinjection in the oocyte of a complex of the invention or of a composition comprising a complex of the invention.

[0275] In some instances the method comprises at least two separate microinjections of at least two engineered proteins of the invention (or two complexes or compositions, polynucleotides, vectors or viral vectors). The invention also provides a method of assisted reproduction, for example in vitro fertilisation, comprising administering any one or more of the engineered proteins, compositions or complexes or polynucleotides, vectors or viral vectors of the invention to the oocyte. Preferences for the administration are as described elsewhere herein.

[0276] The invention also provides an engineered protein according to the invention, a complex according to the invention or a composition according to the invention or a polynucleotide of the invention, a vector of the invention or a viral vector of the invention for use in a method of assisted reproduction, for example for used in a method of in vitro fertilisation.

[0277] The invention provides the use of an engineered protein according to the invention, a complex according to the invention or a composition according to the invention or a polynucleotide of the invention, a vector of the invention or a viral vector of the invention for the manufacture of a medicament for the therapeutic and / or prophylactic treatment of infertility, for example for the manufacture of a medicament for in vitro fertilisation.

[0278] The invention also provides a cell comprising any one or more of the engineered proteins, complexes or compositions, or a polynucleotide of the invention, a vector of the invention or a viral vector according to the invention.

[0279] The invention also provides an oocyte comprising any one or more of the engineered proteins, complexes or compositions or a polynucleotide of the invention, a vector of the invention or a viral vector according to the invention.

[0280] The invention also provides a kit comprising at least a first engineered protein of the invention, for example as set out under Turbo-Linker, and a bridging protein. Preferences for the engineered protein and bridging protein are as set out elsewhere herein.

[0281] The invention also comprises a kit comprising any two or more engineered proteins of the invention and / or complexes of the invention, for example the kit may comprise any one or more of: a) at least 1, 2, 3, 4, 5 or more engineered proteins comprising a first domain that is a nucleic acid binding domain and at least a second domain that is a separase cleavage substrate domain comprising at least one amino acid sequence cleavable by a separase, for example an engineered protein as set out under Anaphase Safeguard described herein; b) at least 1, 2, 3, 4, 5 or more engineered proteins comprising at least a first domain that is a nucleic acid binding domain and at least a second domain that comprises at least one amino acid sequence that recruits at least one phosphatase activity, for example an engineered protein as set out under Anaphase Shield described herein; c) an engineered protein comprising: i) a nucleic acid binding domain, preferably a DNA binding domain; ii) a first interaction domain capable of interacting with a first partner domain; and iii) a second interaction domain capable of interacting with a second partner domain wherein the first interaction domain and second interaction domains are not able to interact with each other within the same protein molecule, for example an engineered protein as set out under Turbo-Linker described herein d) at least 1, 2, 3, 4, 5 or more complexes comprising an engineered protein that comprises: i) a nucleic acid binding domain, preferably a DNA binding domain; ii) a first interaction domain capable of interacting with a first partner domain; and iii) a second interaction domain capable of interacting with a second partner domain wherein the first interaction domain and second interaction domains are not able to interact with each other within the same protein molecule; and / or e) at least 1, 2, 3, 4, 5 or more complexes formed between at least a first engineered protein of the invention, for example as set out under TurboLinker described herein, and a bridging protein.

[0282] In particular embodiments the kit comprises at least one or at least two of any of the engineered proteins as set out herein. For example the kit may comprise at least one or more for example at least two engineered proteins comprising: i) a nucleic acid binding domain, preferably a DNA binding domain; ii) a first interaction domain capable of interacting with a first partner domain; and iii) a second interaction domain capable of interacting with a second partner domain wherein the first interaction domain and second interaction domains are not able to interact with each other within the same protein molecule. for example at least two engineered proteins as set out under Turbo-Linker or Mono-Linker described herein.

[0283] The kit may also comprise a bridging protein as set out herein.

[0284] As mentioned throughout, preferences for features described in relation to one approach set out herein are to be taken as preferences for that feature throughout, unless stated otherwise. For example discussions around the nucleic acid binding domain, interaction domains etc. are to be taken as general discussions that apply to each instance of a nucleic acid binding domain or interaction domain.

[0285] Preferences and options for a given aspect, feature, or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For example, the invention provides: a composition comprising an engineered protein comprising a first domain that is a nucleic acid binding domain and at least a second domain that is a separase cleavage substrate domain comprising at least one amino acid sequence that is recognised and bound by a separase, or that is cleaved by a separase, wherein the nucleic acid binding domain is a TAL DNA binding domain and the at least one amino acid sequence that is recognised and bound by a separase, or that is cleaved by a separase comprises: two sequences that are recognised by the separase binding site from human Securin of SEQ ID NO.5 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.5; and also comprises one sequence that is the separase cleavage site from human Meikin of SEQ ID NO.6 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.6; and also provides: an engineered protein comprising: a) a DNA binding domain that is a ki67 DNA binding domain; b) a first interaction domain capable of interacting with a first partner domain; and c) a second interaction domain capable of interacting with a second partner domain and d) a third interaction domain capable of interacting with a third partner domain where the first interaction domain is a SpyTag domain and the second interaction domain is a DogTag domain and the third interaction domain is a SnoopTag domain; and also provides: a) a first engineered protein comprising: i) a DNA binding domain that is a TAL DNA binding domain of SEQ ID NO. l or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. l; ii) a separase cleavage substrate domain comprising of at least one amino acid sequence that is recognized and bound by a separase, or that is cleaved by a separase of SEQ ID NO.6 or an an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.6; or, a phosphatase or phosphatase-recruiting domain of at least one amino acid sequence of SEQ ID NO.12 or an an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.12; and, iii) a first interaction domain capable of interacting with a first partner domain; and, iv) a second interaction domain capable of interacting with a second partner domain; and, v) a third interaction domain capable of interacting with a third partner domain; and, b) a second engineered protein comprising: i) a DNA binding domain that is a TAL DNA binding domain of SEQ ID NO. l or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. l; and either, ii) a separase cleavage substrate domain comprising of at least one amino acid sequence that is recognized and bound by a separase, or that is cleaved by a separase of SEQ ID NO.6 or an an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.6; and iii) a first partner domain capable of interacting with a first interaction domain; and, iv) a second partner domain capable of interacting with a second interaction domain; and, v) a third partner domain capable of interacting with a third interaction domain; and also provides: a) a first engineered protein comprising: i) a DNA binding domain that is a TAL DNA binding domain of SEQ ID NO. l or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. l; ii) a phosphatase or phosphatase-recruiting domain of at least one amino acid sequence of SEQ ID NO.12 or an an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.12; and, iii) a first interaction domain capable of interacting with a first partner domain; and, iv) a second interaction domain capable of interacting with a second partner domain; and, v) a third interaction domain capable of interacting with a third partner domain; and, b) a second engineered protein comprising: i) a DNA binding domain that is a TAL DNA binding domain of SEQ ID NO. l or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. l; and either, ii) a phosphatase or phosphatase-recruiting domain of at least one amino acid sequence of SEQ ID NO.12 or an an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.12; and, iii) a first partner domain capable of interacting with a first interaction domain; and, iv) a second partner domain capable of interacting with a second interaction domain; and, v) a third partner domain capable of interacting with a third interaction domain; and also provides: an engineered protein comprising: a) a DNA binding domain that is a TAL DNA binding domain of SEQ ID NO.2 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.2; b) a separase cleavage substrate domain comprising of at least one amino acid sequence that is recognized and bound by a separase, or that is cleaved by a separase of SEQ ID NO.8 or an an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.8; c) a phosphatase or phosphatase-recruiting domain of at least one amino acid sequence of SEQ ID NO.12 or an an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.12;

[0286] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0287] Figure Legends

[0288] Figures 1-4 show exemplary arrangements of the engineered proteins and complexes of the invention, and in some instances exemplary arrangements of the interactions between interaction domains. These figures should not be taken as limiting and other combinations and positions of the domains (for example relative to the nucleic acid binding domain) are contemplated by the invention with the figures intended to simply illustrate to the skilled person the concepts described herein.

[0289] The figures illustrate the approaches with reference to a DNA binding domain or chromatin binding domain, however as set out in the claims and the description the engineered proteins comprise a nucleic acid binding domain that may be a DNA / chromatin binding domain but may be a different nucleic acid binding domain. The domain may also be a domain that binds to a protein that binds to DNA / chromatin.

[0290] Figure 1 - illustration of exemplary means of implementing the "Anaphase Safeguard" approach set out herein. A) engineered protein with a separase cleavage substrate domain that comprises a single amino acid sequence that is capable of being recognised by and bound by, or cleaved by, a separase. B) engineered protein with a separase cleavage substrate domain that comprises multiple amino acid sequences that are capable of being recognised by and bound by, or cleaved by, a separase. The multiple sequences may be the same sequence, or may be different sequences. C), D), E), and F) engineered protein comprising more than one separase cleavage substrate domain.

[0291] Figure 2 - illustration of exemplary means of implementing the "Anaphase Shield" approach set out herein. A) illustrates examples of the engineered protein with a second domain that comprises one amino acid sequence that recruits at least one phosphatase activity, three different amino acid sequences that recruit phosphatase activity, and three sequences that recruit phosphatase activity wherein two of the sequences are the same. B) Illustrates exemplary engineered proteins that comprise both a separase cleavage substrate domain as per the Anaphase SafeGuard approach, and a domain that comprises one or more amino acid sequences that recruits at least one phosphatase activity as per the Anaphase Shield approach.

[0292] Figure 3 - A) illustration of exemplary arrangements of domains of the engineered protein set out under the Turbo-Linker and Mono-Linker approaches. Although the figure only shows the DNA binding domain (or nucleic acid binding domain) and the first and second interaction domains, the engineered protein may comprise one or other domains, for example one or more domains as set out herein. B) Illustration of exemplary interactions that are prevented or produced with the engineered proteins as set out in the Turbo-Linker approach, i) demonstrates the inability of the first and second interaction domains to interact with one another within the same protein molecule. Means to prevent this interaction are described in the main text, ii) exemplary arrangement of interactions between two copies of the same engineered protein and genomic DNA. The DNA binding domain of each protein binds to different regions of DNA, and the first and second interaction domains interact with each other in a inter-molecular fashion, resulting in covalently linked engineered protein molecules, tethering the two regions of genomic DNA together.

[0293] Figure 4 - Illustration of exemplary arrangements for putting the Mono-Linker approach into effect. A) a complex is formed between an engineered protein of the invention and a bridging protein, such that one of the domains of the bridging protein is covalently linked to the engineered protein, and one of the domains of the bridging protein and the engineered protein remain unreacted and do not interact with each other. This is an example of the complex in a composition prior to administration to a cell such as an oocyte. Means to prevent these two domains from interacting with one another are described in the main text, and include utilising a low pH, such as a pH of 5 or below. B) upon administration to the cell such as an oocyte, whatever means that were used to prevent the interaction of the two domains in the composition, such as low pH, are alleviated, for instance the pH within the oocyte is around pH7. Once the means of preventing the interaction have been alleviated, the first interaction domain is able to interact with the first partner domain of the bridging protein. At the same time, the DNA binding domains engage with the genomic DNA in the cell (e.g. oocyte), tethering regions of DNA together. The dashed lines illustrate a further molecule of the complex which can interact the complex that has formed between the DNA and two molecules of the complex, demonstrating the ability for a single administration of a pre-formed complex into a cell such as an oocyte to initiate a polymeric network of protein complexes and DNA. C) a complex similar to the aforementioned Mono-Linker, but containing two or more First interaction domains, both prevented from binding the First partner domain at low pH, such as a pH of 5 or below. D) upon administration to the cell such as an oocyte, whatever means that were used to prevent the interaction of the two domains in the composition, such as low pH, are alleviated, for instance the pH within the oocyte is around pH7. Once the means of preventing the interaction have been alleviated, the one of the two First interaction domains is able to interact with the First partner domain of the bridging protein.

[0294] Figure 5 - Chromosome segregation error types arising in the first meiotic and second meiotic divisions of human oocyte maturation. (A) Chromosome segregation patterns that result in gain of an extra chromosome copy. First column from left: correct chromosome segregation leading to a haploid contribution of chromosomes from the egg. Second and third columns: examples of premature separation of sister chromatids (PSSC) errors leading to a gain of an extra chromosome copy; second column illustrates a meiosis I-type PSSC error, and; third column illustrates a meiosis Il-type PSSC error. Fourth column: example of a non-disjunction error resulting in a gain of an extra chromosome copy. Asterisks indicate anomalies relative to correct chromosome segregation. Note that a meiosis Il-type error (third column) is distinguished from non-disjunction (fourth column) in that the chromosome is predivided by the first meiotic division, ultimately failing to segregate correctly in the second meiotic division. (B) Chromosome inheritance outcome of each scenario depicted in (A). Aneuploidy rates in human eggs vary by age. For example, the proportion of aneuploidy meiosis-II human oocytes in different female age groups are shown in Figure IB of Gruhn et al., 2019, PMID: 31604276. Figure 6 - Design and composition of mouse Anaphase Safeguard and variants. (A) Mouse Ki67 C-terminal chromatin binding domain (CBD) (aa: 2873-3173) containing a putative NLS sequence [...PVPEKKRAASSKRH...] indicated by a grey box is fused to monomeric enhanced green fluorescent protein (mEGFP; aa: 2-239). (B) Mouse Ki67 CBD fused to a mouse securin fragment (aa: 99-143) and to monomeric enhanced green fluorescent protein. The securin domain contains an LPE separase pseudobinding site indicated by a grey box, and to monomeric enhanced green fluorescent protein (mEGFP; aa: 2-239). (C) Anaphase SafeGuard: Mouse Ki67 CBD fused to a mouse Rec8 fragment (aa: 391-531) and to monomeric enhanced green fluorescent protein (mEGFP; aa: 2-239). The Rec8 domain contains the second and third separase cleavage sites (respectively, C2 [...ETVEEERA...]; and C3 [...EIEVLREA...]) in addition to two tandem LPE separase pseudobinding sites [...LPELPE...] indicated by grey boxes. (D) Mouse Ki67 CBD fused to a mouse Meikin fragment (aa: 130-189) and to monomeric enhanced green fluorescent protein. The Meikin domain contains a separase cleavage site [...SPELFR...] indicated by a grey box. (E) Anaphase SafeGuard phosphomimic variant: Mouse Ki67 CBD fused to a mouse Rec8 fragment (aa: 391- 531) and to monomeric enhanced green fluorescent protein (mEGFP; aa: 2-239) as in (C). Here, the Rec8 domain is modified whereby 18 serine (S) and threonine (R) amino acid residues are mutated to aspartic acid (D) mimicking the negative charge and size of phosphorylated residues (indicated in sequences 23 and 24). For all construct maps, domains are not drawn to scale. (F) Mouse Ki67 CBD fused to a mouse Rad21 fragment (aa: 376-548) and to monomeric enhanced green fluorescent protein. The Rad21 domain contains four separase cleavage sites (C1-[...LVPEDLRK...]; C2 [...ENPEVPRE...] ; C3 [...EPIIEEPSRL...] and C4 [...DSVMEASR...]) and a single LPE site indicated by grey boxes.

[0295] Figure 7 - AnaphaseSafeguard localizes to chromosomes in mouse oocytes. (A) Representative super-resolution immune-fluorescence imaging of the first meiotic spindle in a metaphase I-stage mouse oocyte. Anaphase Safeguard (green) localizes to bivalent chromosomes (blue) aligned on the first meiotic spindle (grey). Kinetochores indicated in magenta. Boxed regions (left panel) are magnified in the right panel. Scale bars: 2 pm. (B) Representative super-resolution immune- fluorescence imaging of the first meiotic spindle in a metaphase II-stage mouse oocyte. Anaphase Safeguard (green) localizes to chromosomes (blue) aligned on the first meiotic spindle (grey). Kinetochores indicated in magenta. Boxed regions (left panel) are magnified in the right panel. Scale bars: 2 pm. (C) Titration of mRNA coding Anaphase SafeGuard (Figure 6C). Three dilutions of 0.5, 0.05, and 0.005 femtomoles per microliter (fmol / pL) of mouse AnaphaseSafeguard mRNA were run in parallel on a 1% agarose gel. The predicted in vitro transcribed mRNA for mouse AnaphaseSafeguard is 2387 nucleotides (nt) in length. (Left lane) a standard of 10 pL RiboRuler High Range RNA ladder (ThermoScientific) was run in parallel with mRNA.

[0296] Figure 8 - Anaphase Safeguard reduces the frequency of chromosome errors in aged mouse eggs. (A) Representative metaphase II spindles (grey) of young, aged, and aged treated mouse eggs collected from super-resolution immune-fluorescence confocal microscopy. Aged treated eggs were microinjected with 0.05 fmol / pL AnaphaseSafeguard mRNA (green). Kinetochores indicated in magenta. Yellow arrows indicate chromatids resulting from a premature separation of sister chromatids (PSSC) error. Scale bar: 2 pm. (B) Quantification of PSSC error frequency in young (hollow bar) and aged mouse eggs (filled bar), and aged mouse eggs treated with 0.05 fmol / pL of mRNA coding Anaphase Safeguard and variants depicted in figure 2 (grey bars). Percent of PSSC errors for each condition are indicated above the corresponding bars. N, number of analysed oocytes, and; n, number of analysed kinetochore pairs are indicated to the right. Significance testing was performed using a one-sided Fisher's exact test.

[0297] Figure 9 - Anaphase Safeguard increases the number of error-free oocytes from aged mice. Quantification of mouse metaphase II oocytes free of detectable errors (hollow bars) or containing one or more chromosome errors (grey bars). For each condition, the percentage of error-free oocytes is indicated above the hollow bar. Dashed lines indicate the percentage of error-free oocytes in untreated young (upper) and aged (lower) conditions, respectively.

[0298] Figure 10 - Design and composition of human Anaphase Safeguard for mRNA and protein expression. (A) Human Anaphase Safeguard designed for mRNA-based expression. N-terminal FLAG-tagged human Ki67 C-terminal chromatin binding domain (CBD) (aa: 2873-3173) fused to a human Rec8 fragment (aa: 350-487) and to monomeric enhanced green fluorescent protein (mEGFP; aa: 2-239). The Ki67 CBD contains a putative NLS sequence [...PASKKQRVA...] indicated by a grey box. Human Rec8 domain contains the second and third separase cleavafe sites (respectively, C2 [...EEERR...]; and C3 [...EIEVPREA...]) in addition to one LPE separase pseudobinding site [...LPE...] indicated by a grey box. (B) Human Anaphase Safeguard designed for protein purification. N-terminal His-tagged human Ki67 CBD similar to the design in (A). (C) Non-reducing SDS PAGE gel demonstrating nickel-affinity purified Anaphase Safeguard indicated in (B). Predicted molecular weight: 79.5 kDa. Predicted isoelectric point (pl): 7.65.

[0299] Figure 11 - Human Anaphase Safeguard protein localization in live mouse metaphase II eggs. (A) Purified human Anaphase Safeguard protein injected into mouse metaphase II eggs (mEGFP; magenta) co-localizes with chromosomes (SiR-DNA; cyan) (upper panel) compared to signal in protein non-injected eggs (lower panel). Differential Interference Contrast (DIC) in greyscale. Maximum projection of seven Z- sections representing thickness of 10.5 pm. Scale bars: 10 pm. (B) Purified human Anaphase Safeguard protein injected into mouse metaphase II eggs (mEGFP; magenta) co-localizes with chromosomes (SiR-DNA; cyan) (upper panel) compared to signal in protein non-injected eggs (lower panel). Differential Interference Contrast (DIC) in greyscale. Micrographs represent a single z-section 2 pm in thickness. Scale bars: 5 pm.

[0300] Figure 12 - Human Anaphase Safeguard protein localises to chromosomes in live human metaphase I oocytes. (A) Purified human Anaphase Safeguard protein injected into human metaphase I oocytes (mEGFP; magenta) co-localizes with chromosomes (SiR-DNA; cyan) (upper panel) compared to signal in protein non-injected eggs (lower panel). Differential Interference Contrast (DIC) in greyscale. Orthoganal Maximum projection of seven Z-sections representing thickness of 10.5 pm. Scale bars: 10 pm. (B) Purified human Anaphase Safeguard protein injected into human metaphase II eggs (mEGFP; magenta) co-localizes with chromosomes (SiR-DNA; cyan) (upper panel) compared to signal in protein non-injected eggs (lower panel. Differential Interference Contrast (DIC) in greyscale. Micrographs represent a single z-section 2 pm in thickness. Scale bars: 5 pm.

[0301] Figure 13 - Design and composition of mouse Anaphase Shield and variants. (A) N- terminal FLAG-tagged Transcription Activator-Like (TAL) DNA binding domain (DBD) (aa: 1-695) targeting the mouse centromeric repetitive sequence [TGCCATATTCCACGT SEQ ID NO: 129] fused to monomeric enhanced green fluorescent protein (mEGFP; aa: 2-239) and to three identical PP2A- recruiting fragments of mouse Shugoshin-2 (Sgo2CC; aa: 46-91) forming 3xmmSgo2CC. (B) N-terminal FLAG-tagged TAL DBD as in (A) fused to monomeric enhanced green fluorescent protein (mEGFP; aa: 2-239) and to three identical PP2A-recruiting fragments of mouse Striatin-3 (STRN3CC; aa: 86-123) forming 3xmmSTRN3CC. (C) N-terminal FLAG-tagged TAL DBD as in (A) and (B) fused to monomeric enhanced green fluorescent protein (mEGFP; aa: 2-239) and to three identical PP2A-recruiting fragments of mouse Shugoshin-1 (Sgo2CC; aa: 51- 96) forming 3xmmSgolCC.

[0302] Figure 14 - Anaphase Shield localizes to chromosomes in mouse oocytes (A) Representative super-resolution immune-fluorescence imaging of the first meiotic spindle in a metaphase I-stage mouse oocyte. Anaphase Shield (green) localizes to pericentromeric regions of bivalent chromosomes (blue) aligned on the first meiotic spindle (grey). Kinetochores indicated in magenta. Boxed regions (left panel) are magnified in the right panel. Scale bars: 2 pm. (B) Representative super-resolution immune-fluorescence imaging of the first meiotic spindle in a metaphase II-stage mouse oocyte. Anaphase Safeguard (green) localizes to pericentromeric regions of chromosomes (blue) aligned on the first meiotic spindle (grey). Kinetochores indicated in magenta. Boxed regions (left panel) are magnified in the right panel. Scale bars: 2 pm.

[0303] Figure 15 - Anaphase Shield reduces the frequency of chromosome errors in aged mouse eggs. Quantification of PSSC error frequency in young (hollow bar) and aged mouse eggs (filled bar), and aged mouse eggs treated with 0.05 fmol / pL of mRNA coding Anaphase Shield and variants depicted in figure 9 (grey bars). Percent of PSSC errors for each condition are indicated above the corresponding bars. N, number of analysed oocytes, and; n, number of analysed kinetochore pairs are indicated at the bottom. Significance testing was performed using a one-sided Fisher's exact test.

[0304] Figure 16 - Anaphase Shield increases the number of error-free oocytes from aged mice. Quantification of mouse metaphase II oocytes free of detectable errors (hollow bars) or containing one or more chromosome errors (grey bars). For each condition, the percentage of error-free oocytes is indicated above the hollow bar. Dashed lines indicate the percentage of error-free oocytes in untreated young (upper) and aged (lower) conditions, respectively. N, number of analysed oocytes, are indicated at the bottom.

[0305] Figure 17 - Design and composition of human Anaphase Shield and variants. (A) N- terminal FLAG-tagged Transcription Activator-Like (TAL) DNA binding domain (DBD) (aa: 1-695) targeting the human centromeric repetitive sequence [TCCATTCCATTCCATTC] fused to monomeric enhanced green fluorescent protein (mEGFP; aa: 2-239) and to three identical PP2A-recruiting fragments of human Shugoshin-2 (Sgo2CC; aa: 48-94) forming 3xhsSgo2CC. (B) N-terminal FLAG-tagged TAL DBD as in (A) fused to monomeric enhanced green fluorescent protein (mEGFP; aa: 2-239) and to three identical PP2A-recruiting fragments of human Striatin-3 (STRN3CC; aa: 86-123) forming 3xhsSTRN3CC. (C) N-terminal FLAG-tagged TAL DBD as in (A) and (B) fused to monomeric enhanced green fluorescent protein (mEGFP; aa: 2-239) and to three identical PP2A-recruiting fragments of human Shugoshin-1 (Sgo2CC; aa: 51-96) forming 3xhsSgolCC.

[0306] Examples

[0307] Example 1 - Anaphase Safeguard - engineered proteins comprising separase cleavage / binding domains protect oocytes against PSSC

[0308] Figure 6 shows constructs used in this study. The constructs use the mouse Ki67 C- terminal chromatin binding domain to tether the protein to the chromatin, and a series of different separase cleavage domains. The chromatin binding domain is considered to be interchangeable with other DNA or chromatin binding domains.

[0309] Construct A is a control and comprises the chromatin binding domain fused to the reporter GFP. In B the chromatin binding domain is fused to a fragment of mouse securin which comprises 1 LPE motif. In C the chromatin binding domain is fused to a fragment of mouse Rec8 which comprises 2 separase cleavage sites ETVEEERA [SEQ ID NO: 126] and EIEVLREA [SEQ ID NO: 127] and two tandem LPE motifs (LPELPE). D comprises the chromatin binding domain fused to a meikin fragment that comprises a separase cleave site SPELFR [SEQ ID NO: 128]. E comprises the chromatin binding domain fused to an engineered Rec8 fragment which has been modified to change 18 serine (S) and threonine (R) amino acid residues to aspartic acid (D) mimicking the negative charge and size of phosphorylated residues. The domain comprises 2 separase cleavage sites ETVEEERA [SEQ ID NO: 128] and EIEVLREA [SEQ ID NO: 127] and two tandem LPE motifs (LPELPE; SEQ ID NO: 124).

[0310] Figure 7 shows appropriate localisations of the constructs to bivalent chromosomes (blue) aligned on the first meiotic spindle (A) and to chromosomes (B) aligned on the first meiotic spindle (grey). Kinetochores indicated in magenta.

[0311] Figure 8 demonstrates that Anaphase Safeguard reduces the frequency of chromosome errors in aged mouse eggs. Injection of each of constructs C and E reduced the frequency of PSSC errors in mouse metaphase II oocytes, demonstrating that fusion of a separase cleavage substrate to a chromatin binding domain is capable of protecting endogenous cohesin from incorrect separase cleavage in the first meiotic division. The effect is pronounced to the level where with some constructs the error frequency is reduced down close to that of "young" oocytes not exposed to reproductive aging effects.

[0312] Conversely, construct A containing a chromatin binding domain and GFP but lacking a separase cleavage substrate (as defined herein), or construct B containing a separase inhibitory motif not recognised by separase during anaphase, does not provide significant protective against PSSC errors in aged mouse eggs (B).

[0313] Figure 9 looks at the actual number of error-free oocytes, rather than error frequency, and is a key parameter since an aim of the invention is to provide more oocytes in which no errors occur. Injection of oocytes with constructs C and E show a large reduction in the number of aged oocytes that comprise any error at all. Construct E, in which residues have been substituted to mimic phosphorylated residues, shows an increased improvement over that with the wild-type sequence, increasing the proportion of oocytes with no errors at all to the level seen in the young oocytes.

[0314] Figure 10 shows constructs designed to have human-based sequences. The Human Rec8 domain used contains the second and third separase cleavage sites (respectively, C2 [...EEERR...]; and C3 [...EIEVPREA...]) in addition to one LPE separase pseudobinding site [...LPE...] indicated by a grey box. As for the engineered proteins that comprise mouse sequences, the human sequence version of the engineered protein also localises to bivalent chromosomes and chromosomes aligned on the second meiotic spindle (Figure 11 A and B).).

[0315] The human sequence version of the engineered protein localises to bivalent chromosomes in human oocytes. (Figure 12 A and B).

[0316] Example 2 - Anaphase Shield reduces the frequency of chromosome errors in aged mouse eggs

[0317] As set out elsewhere, the premise behind the Anaphase Shield approach involves modulating the phosphorylation status of cohesin. The phosphorylation of cohesin plays a critical regulatory role in its cleavage by separase during cell division, affecting its stability and the timing of its cleavage, which is essential for accurate chromosome segregation. The approach involves stimulating the recruitment of phosphatase activity to the chromatids protects cohesin from cleavage by separase. The recruitment of the phosphatase activity may be a PP2A activity, or may be another phosphatase activity. Figure 13 describes constructs used in the experimental work. The Transcription Activator-Like (TAL) DNA binding domain (DBD) (aa: 1-695) domain [SEQ ID NO: 72] was used in all of constructs A, B and C to target the engineered protein to the chromatin. An N-terminal FLAG tag was used to aid in purification / visualisation but is not considered to be essential to the function of the engineered protein. The DNA binding domain targets the centromeric repetitive sequence [TGCCATATTCCACGT] SEQ ID NO: 129]. The engineered protein involves a fusion to monomeric enhanced green fluorescent protein (mEGFP; aa: 2-239) to aid in visualisation of the protein. As for the tag, the fluorescent reporter protein is not considered to be essential and is present for experimental purposes. The constructs A B and C differ in the protein sequences used to effect phosphatase activity.

[0318] In construct A the protein includes three identical PP2A-recruiting fragments of mouse Shugoshin-2 (Sgo2CC; aa: 46-91) forming 3xmmSgo2CC.

[0319] In construct B, instead of three identical PP2A-recruiting fragments of mouse Shugoshin-2 (Sgo2CC; aa: 46-91), construct B includes three identical PP2A-recruiting fragments of mouse Striatin-3 (STRN3CC; aa: 86-123) forming 3xmmSTRN3CC.

[0320] Similarly for construct C, the engineered protein comprises three identical PP2A- recruiting fragments of mouse Shugoshin-1 (Sgo2CC; aa: 51-96) forming 3xmmSgolCC.

[0321] Figure 14 shows that the engineered proteins localise to the pericentromeric regions of bivalent chromosomes (A) and chromosomes (B) aligned on the first meiotic spindle, as expected.

[0322] Figure 15 demonstrates that the use of the engineered protein that comprises the Sgo2CC domain (construct A) and the engineered protein that comprises the STRN3CC domain (construct B) both reduce the PSSC error frequency in aged oocytes by 70% and 60% respectively. Figure 16 shows that the number of entirely error free aged oocytes increases by 30% (construct A) and 14% (construct B). Figure 16 looks at the actual number of error-free oocytes, rather than error frequency, and is a key parameter since an aim of the invention is to provide more oocytes in which no errors occur. Constructs A and B both increased the proportion of error free oocytes compared to the non-injected oocytes. Figure 17 sets out engineered protein constructs that comprise human sequences, with human Sgo2CC (construct A); human STRN3cc (construct B) and human sgolCC (construct C).

[0323] Example 3 - combination of Anaphase Safeguard and Anaphase Shield

[0324] Both the Anaphase Safeguard approach (which involves the use of an engineered protein that comprises one or more separase binding and / or cleavage sites fused to a DNA / chromatin binding domain) and the Anaphase Shield approach (which targets to the chromatin a domain which aids in phosphorylation of cohesin) reduce the overall frequency of sister chromatid segregation errors and increases the number of oocytes which have no errors at all.

[0325] Figure 21 sets out constructs designed to test both approaches. The constructs comprise a tag (3xFLAG) to aid in purification / imaging, and an eGFP domain to aid in imaging - both of which are not considered essential to the therapeutic effect of the engineered protein. The constructs use the TAL centromeric DNA binding domain (mouse in construct A and human in construct B), though other DNA or chromatin binding domains are expected to also function, as set out elsewhere herein, for example the ki67 domain. The constructs also use the mouse or human Sgo2CC domain as used in Example 3. Also as shown in Example 3, other domains are suitable and function to reduce error frequency / increase error-free oocytes.

[0326] Example 4 - Approach, and Materials & Methods

[0327] Cohesins are typically removed from chromosomes by separase stepwise, with cohesins between homologous chromosomes and cohesins in centromeric regions cleaved prior to the cohesins between sister chromatids. During maternal ageing, cohesins are thought to be lost from both homologous chromosomes and sister chromatids. This could result in the cohesin that holds sister chromatid together being prematurely cleaved prior to anaphase II, leading to PSSC. Based on this, we developed a therapeutic strategy called Anaphase Safeguard, which consists of a chromatin binding domain fused to a separase cleavage substrate (SOS) (Fig. 1). We propose that providing an excess of locally concentrated SOS effectively provides a separase decoy, protecting sister chromatid cohesin from aberrant removal by separase.

[0328] To assess the efficacy of engineered recombinant protein therapeutic candidates (therapeutics), we compared aged oocytes treated with mRNA encoding the therapeutics to untreated aged oocytes (aged controls) and to untreated oocytes from young mice (young controls). The mRNA encoding the therapeutics were not administered to young oocytes as PSSC errors are already low in oocytes from young mice with no impact following treatment. For each engineered recombinant protein therapeutic candidate, efficacy was assessed by quantifying PSSC errors in mouse metaphase II oocytes. In metaphase II, chromosomes that have prematurely separated are highly predisposed to segregation errors in the subsequent divisions causing aneuploidy. However, at metaphase II, chromosomes that have prematurely separated will still be located within the oocyte since second polar body extrusion can only occur from anaphase II following fertilisation. As a result, prematurely separated chromosomes cannot be detected in metaphase II oocytes through genetic assessments for aneuploidy such as PGT-A (Preimplantation Genetic Testing for Aneuploidy). Therefore, quantification of PSSC provides a direct and early indication of error rates in subsequent cell divisions that lead to aneuploidy. The number of PSSC errors varies between oocytes, ranging from no affected chromosomes to single and multiple (complex) errors within the same cell. Complex errors are more commonly observed with advancing maternal age. To more accurately represent therapeutic efficacy, we assessed the reduction in errors on a per chromosome basis, as oocytes with higher initial error rates may show less apparent overall improvement.

[0329] In oocytes from aged mice, treatment with several therapeutic candidates resulted in a statistically significant reduction in the frequency of PSSC errors compared to uninjected controls. Several therapeutic candidates were generated, including a mouse Rec8 protein fragment consisting of amino acids 391 to 531 fused to the chromatin binding domain of mouse Ki67 protein, generating mmKi67-mmRec8-mEGFP, or Anaphase Safeguard. Microinjection of mRNA coding for Anaphase Safeguard into aged mouse oocytes resulted in a 6.39-fold decrease in PSSC relative to rates measured in untreated aged and young oocytes. In practical terms, approximately 1 in 40 chromosomes experienced PSSC errors in untreated oocytes from aged mice; an error rate sufficient to cause sub-fertility and infertility. Following treatment with Anaphase Safeguard, only 1 in 333 chromosomes from aged oocytes underwent a PSSC error. Notably, the frequency of PSSC errors in treated aged oocytes approached levels observed in oocytes from young uninjected controls (0.3% and 0.41%, respectively). This effect was not observed when mRNA coding Ki67 chromatin binding domain (CBD) alone, or Ki67-CBD fused to a fragment of mouse securin was microinjected into aged mouse oocytes. Together, these observations identify that by localising protein fragments that are targeted by separase to chromosomes can significantly reduce PSSC errors in aged mammalian oocytes. Prior to proteolytic cleavage, protein substrates targeted by separase require phosphorylation. We reasoned that by eliminating phosphorylation as a prerequisite for separase binding and cleavage, therapeutic effects to divert separase from targeting endogenous cohesin complexes might be enhanced. Aspartic acid residues can mimic the negative charge and size of phosphorylated serine and threonine residues. We therefore substituted all serine and threonine residues of the mouse Rec8 separase cleavage substrate to aspartic acids, creating the phosphomimic candidate, mmKi67-Rec8S / T>D-mEGFP. Indeed, the phosphomimic candidate demonstrated a more than 35-fold reduction in PSSC error rates relative to untreated aged and young mouse oocytes (Figure 8B). This finding indicates that the therapeutic effects of localising separase substrates to chromosomes can be enhanced by accommodating the charge and shape expected of residues phosphorylated by accessory kinases that control separase substrate recognition and cleavage.

[0330] Incorrect inheritance of even a single chromatid copy is sufficient to block embryo development. The primary aim of the presented therapeutic approach is to increase the number of oocytes that are completely free of errors. Therefore, we quantified the proportion of oocytes with no detectable chromosome error to highlight the therapeutic's impact at generating healthy fertilisable oocytes from aged mice. The proportion of error-free oocytes was increased in the treated aged group, showing a multiple-fold improvement over untreated aged control. Specifically, 85.7% and 95.5% of aged oocytes that received treatment were free of errors, compared to 66.1% in the untreated aged group, and more closely resembling (or even exceeding) the 93.6% observed in young controls. Statistical comparisons of PSSC rates were performed using a one-sided Fisher's exact test and variance measured by a 95% confidence interval with one degree of freedom.

[0331] Research Animals

[0332] Maintenance and handling of mice was carried out by the Biomedical Services at the University of Oxford. C57BL / 6J female mice were purchased from Charles River Laboratories. Young mice were aged between 2 to 4 months, and aged mice were aged between 15.5 to 20.5 months of age. Upon delivery, all mice were acclimated to the animal facility for at least four days prior to use. Mice were housed within appropriate refinement conditions under a 12-hour light / dark cycle, and ad libitum access to food and water. All procedures involving mice were conducted at the animal facility under relevant and required approval and accreditation by the UK Home Office. Culture of Human Oocytes

[0333] Research on human oocytes was approved by the NHS Solihull Research Ethics Committee (25 / WM / 0092). Informed consent for donation of immature human oocytes to research was received from patients undergoing intracytoplasmic sperm injection (ICSI) treatment at Care Fertility Leeds. The oocytes used for research were unsuitable for the patient's treatment and would otherwise have been disposed of.

[0334] I Immature oocytes were vitrified after 42 hours post maturation trigger medication treatment according to standard clinical embryology procedures and stored at -192C. Ocytes were thawed using the Kitazato Warming Kit (Kitazato; VT602) in accordance with the manufacturer's instructions. Following thawing, oocytes were cultured using G-MOPS Plus (Vitrolife; 10130) under NidOil (Nidacon; NO-100) in a non-CO2 incubator.

[0335] Culture of mouse oocytes

[0336] GV-stage oocytes were isolated from ovaries and cultured in homemade M2 medium supplemented with 250 pM dibutyryl cyclic AMP (dbcAMP, Tocris; 1141) under paraffin oil (NidOil, Hunter Scientific; NO-100) (Mogessie, 2020). Only oocytes ~ 70 pm in diameter with centered nuclei and tick zona pell ucida (~ 5 pm) were selected for study.

[0337] Plasmid generation and mRNA synthesis

[0338] DNA sequences were synthesised as fragments (Genscript) and subcloned into a pGEM- HE vector (V012814; NovoPro Bioscience) using standard restriction enzyme digestion and ligation methods (New England Biolabs). mRNA was in vitro synthesised using the HiScribe T7 ARCA mRNA kit (E2065S; New England Biolabs) following the manufacturer's instructions. mRNA concentration was quantified using a Qubit RNA HS Assay kit (Q32852; Fisher Scientific) and diluted to a concentration of 0.05 fmol / pL prior to injection. HiScribe T7 ARCA mRNA kit (E2065S; New England Biolabs) following the manufacturer's instructions. mRNA concentration was quantified using a Qubit RNA HS Assay kit (Q32852; Fisher Scientific) and diluted to a concentration of 0.05 fmol / pL prior to injection.

[0339] Protein expression and purification

[0340] The DNA sequence for 6xHis-hsKi67-hsRec8-mEGFP was cloned into the bacterial expression vector pET41a+ using standard molecular biology techniques and confirmed by Sanger sequencing. Small-scale expression screening in E. coli was carried out to identify optimal expression conditions. The plasmid was transformed into chemically competent E. coli expression BL21(DE3) strain and cultured at 37° C in Terrific Broth until an optical density of 0.6 was reached. Expression was induced using 0.1 mM IPTG and grown overnight at 18° C. The bacterial culture was pelleted and resuspended in 1 ml of lysis buffer (25 mM HEPES, 200 mM KCI, pH 7.6, protease inhibitor tablets) and lysed by sonication (70% amplitude, 5 minutes, 30 seconds on / off). Lysates were incubated overnight at 4° C in 1000 pl Ni-magnetic beads equilibrated with 5 mL equilibration buffer (25 mM HEPES, 200 mM KCI, pH 7.6). Beads were washed with wash buffer (25 mM HEPES, 200 mM KCI, pH 7.6). The beads were eluted with equilibration buffer containing either 200 mM imidazole or 300 mM imidazole. Eluted fractions were analysed by SDS-PAGE and absorbance at 280 nm. Eluted fractions were pooled, concentrated and buffer exchanged using Amicon Ultra Centrifugal Filter, 10 kDa MWCO. Purified protein was then dialysed in a buffer containing 25 mM HEPES, 200 mM KCI, pH 7.88.

[0341] Microinjection of oocytes

[0342] All mRNA was diluted to a concentration of 0.05 fmol / pL prior to microinjection. All protein was diluted to a concentration of 0.2 mq / mL prior to microinjection. GV-stage oocytes were microinjected using a Femtojet 5247 and Micro-ePore based microinjection setup. Oocytes were microinjected in small batches to avoid cooling and evaporation of media. After microinjection, oocytes were washed through droplets of fresh M2 medium containing 250 pM dbcAMP under NidOil paraffin oil and incubated at 37° C for three allows to allow for protein expression. Oocytes were then washed into fresh M2 medium without dbcAMP and matured for 16 hours at 37° C.

[0343] Immunofluorescence & Staining

[0344] Oocytes were briefly pre-permeabilised in PBS with 0.25% triton X-100 prior to fixation. Oocytes were incubated at 37°C for 20 minutes in a fixative solution containing 100 mM HEPEs, 50 mM EGTA, 10 mM MgSO4, 2% methanol-free formaldehyde, and 0.2% triton X-100. Fixed oocytes were permeabilised in PBS with 0.5% triton X-100 (PBT) at 4°C for up to 4 days. Oocytes were blocked in PBT supplemented with 5% BSA for up to 5 hours at room temperature. All primary antibodies were incubated at 4°C overnight followed by washes in PBT with 5% BSA for up to 2 hours. All secondary antibodies were incubated at room temperature for 1 hour followed by washes in PBT with 5% BSA for up to 2 hours. DNA was stained with Hoeschst 33342 (H1399; Fisher Scientific, 1 :400) for 1 hour at room temperature followed by washes in PBT with 5% BSA for up to 2 hours. Kinetochores were stained using primary mouse anti-Hecl antibody (sc-515550; Santa Cruz Biosciences, 1 : 100) and Alexa-Fluor-488-labelled secondary anti-mouse antibody (A-21202; Fisher Scientific, 1 :200). GFP was stained using primary rabbit anti-GFP antibody (Ab6556; Abeam, 1: 100) and Alexa-Fluor-568-labelled secondary anti-rabbit antibody (A- 10042; Fisher Scientific, 1:200). Microtubules were stained using primary rat antitubulin antibody (MCA78G; Biorad, 1 : 100) and Alexa-Fluor-647-labelled secondary anti-rat antibody (A-21247; Fisher Scientific, 1 :200)

[0345] Immunofluorescence confocal microscopy imaging

[0346] Fixed and immunofluorescently-labelled oocytes were imaged in PBS covered with NidOil paraffin oil in a 35 mm glass bottom dish with a No.l thickness coverslip (Cellvis). Microscopy was performed using LSM-980 laser scanning confocal microscopes (Zeiss) equipped with a 40x C-apochromat 1.2 NA water-immersion objective (Zeiss). Super-resolution images of fixed immunofluorescently-labelled oocytes were acquired using the Airyscan module (Zeiss) and processed using ZEN (Zeiss) software after acquisition. For each oocyte, 100-120150 z-sections were acquired to ensure all chromosomes within the meiotic spindle were detected. Care was taken with laser power, pixel-dwell time and detector gain to prevent photobleaching or saturation.

[0347] Live confocal imaging

[0348] Mouse oocytes were washed into nine times 20 pL droplets of M2 media containing 250 nM SiR-DNA (Spyrochrome) to label chromosomes and incubated for one hour before transfer and imaging in a Zeiss LSM980 confocal microscope equipped with an incubated chamber set to 37°C. The procedure was identical for live imaging of human oocytes, except human oocytes were washed into nine times 20pL droplets of GMOPS Plus containing 250 nM SiR-DNA. For wide field of view images, 20 z-sections were acquired with a pinhole corresponding to a 3 pm z-section thickness with a z-step of 1.5 pm and two-times averaging. For narrow field of view images, 20 z-sections were acquired with a pinhole corresponding to a 2 pm z-section thickness with a z-step of 1.0 pm and two-times averaging.

[0349] Data analysis and quantification

[0350] Images of meiotic spindles and chromosomes in oocytes were imaged by superresolution AiryScan microscopy and analysed using Imaris (Bitplane) software. Three- dimensional reconstructions of chromosomes were generated using the 'Surface' tool of Imaris for the 405 nm channel corresponding to Hoescht stain (DNA). The generated chromosome surfaces were then used to segment signal proximal to DNA in the 488 nm channel corresponding to kinetochores signal (Heel; NDC80). Segmentation was necessary in cases of high observed background of antibody signal. For those oocytes included in the study, three-dimensional reconstructions of sister kinetochore pairs of metaphase-II chromosomes were generated using the semiautomated 'Spot' detection tool in Imaris for the 488 nm channel corresponding to kinetochores labels. The distance between sister kinetochore pairs was then measured using the 'Measurements point' function of Imaris, where the distance measured corresponded to the interkinetochore distance (iKt) from the centre of each sister kinetochore spot pair.

[0351] PSSC was defined by any interkinetochore distance greater than or equal to 4 pm; or where sister chromosomes were visibly disjoined; and / or where sister chromosomes were aligned perpendicular to the spindle axis; and / or sister chromosomes were localized at the extremes of the spindle poles. When chromosomes or chromatids were undetected and missing, they were counted as 'aneuploid'. Equally, if extra chromatids or chromosomes were detected they were also counted as 'aneuploid'. Extra or missing chromatids / chromosomes were documented and excluded from total chromosome counts when analysing PSSC, as they correspond to a different error type that should be analysed independently or included if total chromosome errors were analysed. In total, only one instance of 'aneuploidy' was detected in the present study.

[0352] Statistical analysis

[0353] For the chromosome error reduction assay, statistical significance was calculated using the one-tailed Fisher's Exact test using GraphPad, where the total number of intact sister chromatids versus the total number of PSSC errors was analyzed. Here, a 95% confidence interval with 1 degree of freedom was used to determine variance in the measurements.

[0354] Sequences

[0355] SEQ ID NO: 1 > Protein sequence for human Ki-67 DNA binding domain (aa2930-3256)

[0356] HTEELANGAADSFTSAPKQTPDSGKPLKISRRVLRAPKVEPVGDVVSTRDPVKSQSKSNTSLPPLPFKRGGGKD GSVTGIKRLRCMPAPEEIVEELPASKKQRVAPRARGKSSEPVVIMKRSLRTSAKRIEPAEELNSNDMKTNKEEH KLQDSVPENKGISLRSRRQNKTEAEQQITEVFVLAERIEINRNEKKPMKTSPEMDIQNPDDGARKPIPRDKVTE NKRCLRSARQNESSQPKVAEESGGQKSAKVLMQNQKGKGEAGNSDSMCLRSRKTKSQPAASTLESKSVQRV TRS VKRC AENPKKAEDNVC VKKIRTRSHRD SEDI

[0357] SEQ ID NO: 2 > Protein sequence for DNA binding alpha-solenoid domain from the TAL01 engineered protein (aal- 789) MADYKDHDGDYKDHDIDYKDDDDKGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL SQHPAALGTVAVTYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGV TAMEAVHASRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETV QRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLL PVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLC QAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHG LTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPA QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVA IASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASH DGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRVA

[0358] The above sequence contains an N-terminal 3x FLAG tag.

[0359] SEQ ID NO: 3 > Protein sequence for human chromo box protein 5 (CBX5; heterochromatin protein 1 alpha) (aal-191) MGKKTKRT AD S S S SEDEEE Y V VEK VLDRR V VKGQ VE YLLKWKGF SEEHNTWEPEKNLD CPELI SEFMKKYK KMKEGENNKPREKSESNKRKSNFSNSADDIKSKKKREQSNDIARGFERGLEPEKIIGATDSCGDLMFLMKWKD TDEADLVLAKEANVKCPQIVIAFYEERLTWHAYPEDAENKEKETAKS

[0360] SEQ ID NO: 4 > Protein sequence for phosphorylated Histone 2A binding domain from human Shugoshin 2 (aa!242- 1265)

[0361] RTSRRRRCTPFYFKEPSLRDKMRR

[0362] SEQ ID NO: 5 > Protein sequence for human Securin separase-binding domain (aa!02-146):

[0363] SSVPASDDAYPEIEKFFPFNPLDFESFDLPEEHQIAHLPLSGVPL

[0364] SEQ ID NO: 6 > Protein sequence for human Meikin separase-cleavage target (aa!25-185)

[0365] ASSLLSYSVTDSYAEYKSFEESFPSPELFRKSDYLDWECPNLEEHMQWKNSTLLDTSKAVA

[0366] SEQ ID NO: 7 > Protein sequence for human Rec8 separase-cleavage target (aa350-487)

[0367] RTPTLSGWLPPELLGLWTHCAQPPPKALRRELPEEAAAEEERRKIEVPSEIEVPREALEPSVPLMVSLEISLEAAE EEKSRISLIPPEERWAWPEVEAPEAPALPVVPELPEVPMEMPLVLPPELELLSLEAVHRAVA

[0368] SEQ ID NO: 8 > Protein sequence for human Rec8 separase-cleavage target (aa385-469) phospho-mimic (S / T to D substitutions):

[0369] RDPDLDGWLPPELLGLWDHCAQPPPKALRRELPEEAAAEEERRKIEVPDEIEVPREALEPDVPLMVDLEIDLEA AEEEKDRIDLIPPEERWAWPEVEAPEAPALPVVPELPEVPMEMPLVLPPELELLDLEAVHRAVA

[0370] SEQ ID NO: 9 > Protein sequence for the centromeric protein C (CENP-C) binding domain from human Meikin (aa328- 373):

[0371] ANASEICCIIRTSPGTRQVKNKGVIVKKKKYSLPKDTPQDIIIKMA

[0372] SEQ ID NO: 10 > Protein sequence for human Shugoshin 1 PP2A binding site (aa51-96):

[0373] STLLKNYQDNNKMLVLALENEKSKVKEAQDIILQLRKECYYLTCQL

[0374] SEQ ID NO: 11 > Protein sequence for human Shugoshin 2 PP2A binding site (aa49-94): IFKISLKHNNRALAQALSREKENSRRITTEKMLLQKEVEKLNFENT

[0375] SEQ ID NO: 12: > Protein sequence for human Striatin3 PP2A binding site (86-123): WEVERAELQARIAFLQGERKGQENLKKDLVRRIKMLEY SEQ ID NO: 13 > Protein sequence for human PP2A scaffold subunit A alpha (PPP2Aalpha):

[0376] MAAADGDDSLYPIAVLIDELRNEDVQLRLNSIKKLSTIALALGVERTRSELLPFLTDTIYDEDEVLLALAEQLGT

[0377] FTTLVGGPEYVHCLLPPLESLATVEETVVRDKAVESLRAISHEHSPSDLEAHFVPLVKRLAGGDWFTSRTSACG

[0378] LFSVCYPRVSSAVKAELRQYFRNLCSDDTPMVRRAAASKLGEFAKVLELDNVKSEIIPMFSNLASDEQDSVRL

[0379] LAVEACVNIAQLLPQEDLEALVMPTLRQAAEDKSWRVRYMVADKFTELQKAVGPEITKTDLVPAFQNLMKD CEAEVRAAASHKVKEFCENLSADCRENVIMSQILPCIKELVSDANQHVKSALASVIMGLSPILGKDNTIEHLLPL FLAQLKDECPEVRLNIISNLDCVNEVIGIRQLSQSLLPAIVELAEDAKWRVRLAIIEYMPLLAGQLGVEFFDEKL

[0380] NSLCMAWLVDHVYAIREAATSNLKKLVEKFGKEWAHATIIPKVLAMSGDPNYLHRMTTLFCINVLSEVCGQD ITTKHMLPTVLRMAGDPVANVRFNVAKSLQKIGPILDNSTLQSEVKPILEKLTQDQDVDVKYFAQEALTVLSL A

[0381] SEQ ID NO: 14 > Protein sequence for human PP2 A Regulatory subunit B’ gamma (PPP2R5C) (aal-579):

[0382] MPNKNKKEKESPKAGKSGKSSKEGQDTVESEQISVRKNSLVAVPSTVSAKIKVPVSQPIVKKDKRQNSSRFSAS

[0383] NNRELQKLPSLKDVPPADQEKLFIQKLRQCCVLFDFVSDPLSDLKWKEVKRAALSEMVEYITHNRNVIIEPIYP

[0384] EVVHMFAVNMFRTLPPSSNPTGAEFDPEEDEPTLEAAWPHLQLVYEFFLRFLESPDFQPNIAKKYIDQKFVLQL

[0385] LELFDSEDPRERDFLKTTLHRIYGKFLGLRAYIRKQINNIFYRFIYETEHHNGIAELLEILGSIINGFALPLKEEHKI

[0386] FLLKVLLPLHKVKSLSVYHPQLAYCVVQFLEKDSTLTEPVVMALLKYWPKTHSPKEVMFLNELEEILDVIEPSE

[0387] FVKIMEPLFRQLAKCVSSPHFQVAERALYYWNNEYIMSLISDNAAKILPIMFPSLYRNSKTHWNKTIHGLIYNA LKLFMEMNQKLFDDCTQQFKAEKLKEKLKMKEREEAWVKIENLAKANPQYTVYSQASTMSIPVAMETDGPL FEDVQMLRKTVKDEAHQAQKDPKKDRPLARRKSELPQDPHTKKALEAHCRADELASQDGR

[0388] SEQ ID NO: 15 > Protein sequence for human PP2A Catalytic subunit C alpha (PPP2CA) (aal-309):

[0389] MDEKVFTKELDQWIEQLNECKQLSESQVKSLCEKAKEILTKESNVQEVRCPVTVCGDVHGQFHDLMELFRIG GKSPDTNYLFMGDYVDRGYYSVETVTLLVALKVRYRERITILRGNHESRQITQVYGFYDECLRKYGNANVWK YFTDLFDYLPLTALVDGQIFCLHGGLSPSIDTLDHIRALDRLQEVPHEGPMCDLLWSDPDDRGGWGISPRGAG

[0390] YTFGQDISETFNHANGLTLVSRAHQLVMEGYNWCHDRNVVTIFSAPNYCYRCGNQAAIMELDDTLKYSFLQF DPAPRRGEPHVTRRTPDYFL

[0391] SEQ ID NO: 16 > DNA sequence for human Ki-67 full-length:

[0392] ATGTGGCCCACGAGACGCCTGGTTACTATCAAAAGGAGCGGGGTCGACGGTCCCCACTTTCCCCTGAGCC

[0393] TCAGCACCTGCTTGTTTGGAAGGGGTATTGAATGTGACATCCGTATCCAGCTTCCTGTTGTGTCAAAACAA

[0394] CATTGCAAAATTGAAATCCATGAGCAGGAGGCAATATTACATAATTTCAGTTCCACAAATCCAACACAAG

[0395] TAAATGGGTCTGTTATTGATGAGCCTGTACGGCTAAAACATGGAGATGTAATAACTATTATTGATCGTTCC

[0396] TTCAGGTATGAAAATGAAAGTCTTCAGAATGGAAGGAAGTCAACTGAATTTCCAAGAAAAATACGTGAA

[0397] CAGGAGCCAGCACGTCGTGTCTCAAGATCTAGCTTCTCTTCTGACCCTGATGAGAAAGCTCAAGATTCCA

[0398] AGGCCTATTCAAAAATCACTGAAGGAAAAGTTTCAGGAAATCCTCAGGTACATATCAAGAATGTCAAAG

[0399] AAGACAGTACCGCAGATGACTCAAAAGACAGTGTTGCTCAGGGAACAACTAATGTTCATTCCTCAGAACA

[0400] TGCTGGACGTAATGGCAGAAATGCAGCTGATCCCATTTCTGGGGATTTTAAAGAAATTTCCAGCGTTAAA

[0401] TTAGTGAGCCGTTATGGAGAATTGAAGTCTGTTCCCACTACACAATGTCTTGACAATAGCAAAAAAAATG

[0402] AATCTCCCTTTTGGAAGCTTTATGAGTCAGTGAAGAAAGAGTTGGATGTAAAATCACAAAAAGAAAATGT

[0403] CCTACAGTATTGTAGAAAATCTGGATTACAAACTGATTACGCAACAGAGAAAGAAAGTGCTGATGGTTTA

[0404] CAGGGGGAGACCCAACTGTTGGTCTCGCGTAAGTCAAGACCAAAATCTGGTGGGAGCGGCCACGCTGTG

[0405] GCAGAGCCTGCTTCACCTGAACAAGAGCTTGACCAGAACAAGGGGAAGGGAAGAGACGTGGAGTCTGTT

[0406] CAGACTCCCAGCAAGGCTGTGGGCGCCAGCTTTCCTCTCTATGAGCCGGCTAAAATGAAGACCCCTGTAC AATATTCACAGCAACAAAATTCTCCACAAAAACATAAGAACAAAGACCTGTATACTACTGGTAGAAGAG

[0407] AATCTGTGAATCTGGGTAAAAGTGAAGGCTTCAAGGCTGGTGATAAAACTCTTACTCCCAGGAAGCTTTC

[0408] AACTAGAAATCGAACACCAGCTAAAGTTGAAGATGCAGCTGACTCTGCCACTAAGCCAGAAAATCTCTCT

[0409] TCCAAAACCAGAGGAAGTATTCCTACAGATGTGGAAGTTCTGCCTACGGAAACTGAAATTCACAATGAGC

[0410] CATTTTTAACTCTGTGGCTCACTCAAGTTGAGAGGAAGATCCAAAAGGATTCCCTCAGCAAGCCTGAGAA

[0411] ATTGGGCACTACAGCTGGACAGATGTGCTCTGGGTTACCTGGTCTTAGTTCAGTTGATATCAACAACTTTG

[0412] GTGATTCCATTAATGAGAGTGAGGGAATACCTTTGAAAAGAAGGCGTGTGTCCTTTGGTGGGCACCTAAG

[0413] ACCTGAACTATTTGATGAAAACTTGCCTCCTAATACGCCTCTCAAAAGGGGAGAAGCCCCAACCAAAAGA

[0414] AAGTCTCTGGTAATGCACACTCCACCTGTCCTGAAGAAAATCATCAAGGAACAGCCTCAACCATCAGGAA

[0415] AACAAGAGTCAGGTTCAGAAATCCATGTGGAAGTGAAGGCACAAAGCTTGGTTATAAGCCCTCCAGCTCC

[0416] TAGTCCTAGGAAAACTCCAGTTGCCAGTGATCAACGCCGTAGGTCCTGCAAAACAGCCCCTGCTTCCAGC

[0417] AGCAAATCTCAGACAGAGGTTCCTAAGAGAGGAGGGAGAAAGAGTGGCAACCTGCCTTCAAAGAGAGTG

[0418] TCTATCAGCCGAAGTCAACATGATATTTTACAGATGATATGTTCCAAAAGAAGAAGTGGTGCTTCGGAAG

[0419] CAAATCTGATTGTTGCAAAATCATGGGCAGATGTAGTAAAACTTGGTGCAAAACAAACACAAACTAAAG

[0420] TCATAAAACATGGTCCTCAAAGGTCAATGAACAAAAGGCAAAGAAGACCTGCTACTCCAAAGAAGCCTG

[0421] TGGGCGAAGTTCACAGTCAATTTAGTACAGGCCACGCAAACTCTCCTTGTACCATAATAATAGGGAAAGC

[0422] TCATACTGAAAAAGTACATGTGCCTGCTCGACCCTACAGAGTGCTCAACAACTTCATTTCCAACCAAAAA

[0423] ATGGACTTTAAGGAAGATCTTTCAGGAATAGCTGAAATGTTCAAGACCCCAGTGAAGGAGCAACCGCAG

[0424] TTGACAAGCACATGTCACATCGCTATTTCAAATTCAGAGAATTTGCTTGGAAAACAGTTTCAAGGAACTG

[0425] ATTCAGGAGAAGAACCTCTGCTCCCCACCTCAGAGAGTTTTGGAGGAAATGTGTTCTTCAGTGCACAGAA

[0426] TGCAGCAAAACAGCCATCTGATAAATGCTCTGCAAGCCCTCCCTTAAGACGGCAGTGTATTAGAGAAAAT

[0427] GGAAACGTAGCAAAAACGCCCAGGAACACCTACAAAATGACTTCTCTGGAGACAAAAACTTCAGATACT

[0428] GAGACAGAGCCTTCAAAAACAGTATCCACTGCAAACAGGTCAGGAAGGTCTACAGAGTTCAGGAATATA

[0429] CAGAAGCTACCTGTGGAAAGTAAGAGTGAAGAAACAAATACAGAAATTGTTGAGTGCATCCTAAAAAGA

[0430] GGTCAGAAGGCAACACTACTACAACAAAGGAGAGAAGGAGAGATGAAGGAAATAGAAAGACCTTTTGA

[0431] GACATATAAGGAAAATATTGAATTAAAAGAAAACGATGAAAAGATGAAAGCAATGAAGAGATCAAGAA

[0432] CTTGGGGGCAGAAATGTGCACCAATGTCTGACCTGACAGACCTCAAGAGCTTGCCTGATACAGAACTCAT

[0433] GAAAGACACGGCACGTGGCCAGAATCTCCTCCAAACCCAAGATCATGCCAAGGCACCAAAGAGTGAGAA

[0434] AGGCAAAATCACTAAAATGCCCTGCCAGTCATTACAACCAGAACCAATAAACACCCCAACACACACAAA

[0435] ACAACAGTTGAAGGCATCCCTGGGGAAAGTAGGTGTGAAAGAAGAGCTCCTAGCAGTCGGCAAGTTCAC

[0436] ACGGACGTCAGGGGAGACCACGCACACGCACAGAGAGCCAGCAGGAGATGGCAAGAGCATCAGAACGT

[0437] TTAAGGAGTCTCCAAAGCAGATCCTGGACCCAGCAGCCCGTGTAACTGGAATGAAGAAGTGGCCAAGAA

[0438] CGCCTAAGGAAGAGGCCCAGTCACTAGAAGACCTGGCTGGCTTCAAAGAGCTCTTCCAGACACCAGGTCC

[0439] CTCTGAGGAATCAATGACTGATGAGAAAACTACCAAAATAGCCTGCAAATCTCCACCACCAGAATCAGTG

[0440] GACACTCCAACAAGCACAAAGCAATGGCCTAAGAGAAGTCTCAGGAAAGCAGATGTAGAGGAAGAATTC

[0441] TTAGCACTCAGGAAACTAACACCATCAGCAGGGAAAGCCATGCTTACGCCCAAACCAGCAGGAGGTGAT

[0442] GAGAAAGACATTAAAGCATTTATGGGAACTCCAGTGCAGAAACTGGACCTGGCAGGAACTTTACCTGGC

[0443] AGCAAAAGACAGCTACAGACTCCTAAGGAAAAGGCCCAGGCTCTAGAAGACCTGGCTGGCTTTAAAGAG

[0444] CTCTTCCAGACTCCTGGTCACACCGAGGAATTAGTGGCTGCTGGTAAAACCACTAAAATACCCTGCGACT

[0445] CTCCACAGTCAGACCCAGTGGACACCCCAACAAGCACAAAGCAACGACCCAAGAGAAGTATCAGGAAAG

[0446] CAGATGTAGAGGGAGAACTCTTAGCGTGCAGGAATCTAATGCCATCAGCAGGCAAAGCCATGCACACGC

[0447] CTAAACCATCAGTAGGTGAAGAGAAAGACATCATCATATTTGTGGGAACTCCAGTGCAGAAACTGGACCT

[0448] GACAGAGAACTTAACCGGCAGCAAGAGACGGCCACAAACTCCTAAGGAAGAGGCCCAGGCTCTGGAAG

[0449] ACCTGACTGGCTTTAAAGAGCTCTTCCAGACCCCTGGTCATACTGAAGAAGCAGTGGCTGCTGGCAAAAC

[0450] TACTAAAATGCCCTGCGAATCTTCTCCACCAGAATCAGCAGACACCCCAACAAGCACAAGAAGGCAGCC CAAGACACCTTTGGAGAAAAGGGACGTACAGAAGGAGCTCTCAGCCCTGAAGAAGCTCACACAGACATC

[0451] AGGGGAAACCACACACACAGATAAAGTACCAGGAGGTGAGGATAAAAGCATCAACGCGTTTAGGGAAA

[0452] CTGCAAAACAGAAACTGGACCCAGCAGCAAGTGTAACTGGTAGCAAGAGGCACCCAAAAACTAAGGAA

[0453] AAGGCCCAACCCCTAGAAGACCTGGCTGGCTTGAAAGAGCTCTTCCAGACACCAGTATGCACTGACAAGC

[0454] CCACGACTCACGAGAAAACTACCAAAATAGCCTGCAGATCACAACCAGACCCAGTGGACACACCAACAA

[0455] GCTCCAAGCCACAGTCCAAGAGAAGTCTCAGGAAAGTGGACGTAGAAGAAGAATTCTTCGCACTCAGGA

[0456] AACGAACACCATCAGCAGGCAAAGCCATGCACACACCCAAACCAGCAGTAAGTGGTGAGAAAAACATCT

[0457] ACGCATTTATGGGAACTCCAGTGCAGAAACTGGACCTGACAGAGAACTTAACTGGCAGCAAGAGACGGC

[0458] TACAAACTCCTAAGGAAAAGGCCCAGGCTCTAGAAGACCTGGCTGGCTTTAAAGAGCTCTTCCAGACACG

[0459] AGGTCACACTGAGGAATCAATGACTAACGATAAAACTGCCAAAGTAGCCTGCAAATCTTCACAACCAGA

[0460] CCCAGACAAAAACCCAGCAAGCTCCAAGCGACGGCTCAAGACATCCCTGGGGAAAGTGGGCGTGAAAGA

[0461] AGAGCTCCTAGCAGTTGGCAAGCTCACACAGACATCAGGAGAGACTACACACACACACACAGAGCCAAC

[0462] AGGAGATGGTAAGAGCATGAAAGCATTTATGGAGTCTCCAAAGCAGATCTTAGACTCAGCAGCAAGTCT

[0463] AACTGGCAGCAAGAGGCAGCTGAGAACTCCTAAGGGAAAGTCTGAAGTCCCTGAAGACCTGGCCGGCTT

[0464] CATCGAGCTCTTCCAGACACCAAGTCACACTAAGGAATCAATGACTAACGAAAAAACTACCAAAGTATCC

[0465] TACAGAGCTTCACAGCCAGACCTAGTGGACACCCCAACAAGCTCCAAGCCACAGCCCAAGAGAAGTCTC

[0466] AGGAAAGCAGACACTGAAGAAGAATTTTTAGCATTTAGGAAACAAACGCCATCAGCAGGCAAAGCCATG

[0467] CACACACCCAAACCAGCAGTAGGTGAAGAGAAAGACATCAACACGTTTTTGGGAACTCCAGTGCAGAAA

[0468] CTGGACCAGCCAGGAAATTTACCTGGCAGCAATAGACGGCTACAAACTCGTAAGGAAAAGGCCCAGGCT

[0469] CTAGAAGAACTGACTGGCTTCAGAGAGCTTTTCCAGACACCATGCACTGATAACCCCACGACTGATGAGA

[0470] AAACTACCAAAAAAATACTCTGCAAATCTCCGCAATCAGACCCAGCGGACACCCCAACAAACACAAAGC

[0471] AACGGCCCAAGAGAAGCCTCAAGAAAGCAGACGTAGAGGAAGAATTTTTAGCATTCAGGAAACTAACAC

[0472] CATCAGCAGGCAAAGCCATGCACACGCCTAAAGCAGCAGTAGGTGAAGAGAAAGACATCAACACATTTG

[0473] TGGGGACTCCAGTGGAGAAACTGGACCTGCTAGGAAATTTACCTGGCAGCAAGAGACGGCCACAAACTC

[0474] CTAAAGAAAAGGCCAAGGCTCTAGAAGATCTGGCTGGCTTCAAAGAGCTCTTCCAGACACCAGGTCACA

[0475] CTGAGGAATCAATGACCGATGACAAAATCACAGAAGTATCCTGCAAATCTCCACAACCAGACCCAGTCA

[0476] AAACCCCAACAAGCTCCAAGCAACGACTCAAGATATCCTTGGGGAAAGTAGGTGTGAAAGAAGAGGTCC

[0477] TACCAGTCGGCAAGCTCACACAGACGTCAGGGAAGACCACACAGACACACAGAGAGACAGCAGGAGAT

[0478] GGAAAGAGCATCAAAGCGTTTAAGGAATCTGCAAAGCAGATGCTGGACCCAGCAAACTATGGAACTGGG

[0479] ATGGAGAGGTGGCCAAGAACACCTAAGGAAGAGGCCCAATCACTAGAAGACCTGGCCGGCTTCAAAGAG

[0480] CTCTTCCAGACACCAGACCACACTGAGGAATCAACAACTGATGACAAAACTACCAAAATAGCCTGCAAA

[0481] TCTCCACCACCAGAATCAATGGACACTCCAACAAGCACAAGGAGGCGGCCCAAAACACCTTTGGGGAAA

[0482] AGGGATATAGTGGAAGAGCTCTCAGCCCTGAAGCAGCTCACACAGACCACACACACAGACAAAGTACCA

[0483] GGAGATGAGGATAAAGGCATCAACGTGTTCAGGGAAACTGCAAAACAGAAACTGGACCCAGCAGCAAGT

[0484] GTAACTGGTAGCAAGAGGCAGCCAAGAACTCCTAAGGGAAAAGCCCAACCCCTAGAAGACTTGGCTGGC

[0485] TTGAAAGAGCTCTTCCAGACACCAATATGCACTGACAAGCCCACGACTCATGAGAAAACTACCAAAATA

[0486] GCCTGCAGATCTCCACAACCAGACCCAGTGGGTACCCCAACAATCTTCAAGCCACAGTCCAAGAGAAGTC

[0487] TCAGGAAAGCAGACGTAGAGGAAGAATCCTTAGCACTCAGGAAACGAACACCATCAGTAGGGAAAGCTA

[0488] TGGACACACCCAAACCAGCAGGAGGTGATGAGAAAGACATGAAAGCATTTATGGGAACTCCAGTGCAGA

[0489] AATTGGACCTGCCAGGAAATTTACCTGGCAGCAAAAGATGGCCACAAACTCCTAAGGAAAAGGCCCAGG

[0490] CTCTAGAAGACCTGGCTGGCTTCAAAGAGCTCTTCCAGACACCAGGCACTGACAAGCCCACGACTGATGA

[0491] GAAAACTACCAAAATAGCCTGCAAATCTCCACAACCAGACCCAGTGGACACCCCAGCAAGCACAAAGCA

[0492] ACGGCCCAAGAGAAACCTCAGGAAAGCAGACGTAGAGGAAGAATTTTTAGCACTCAGGAAACGAACACC

[0493] ATCAGCAGGCAAAGCCATGGACACACCAAAACCAGCAGTAAGTGATGAGAAAAATATCAACACATTTGT

[0494] GGAAACTCCAGTGCAGAAACTGGACCTGCTAGGAAATTTACCTGGCAGCAAGAGACAGCCACAGACTCC TAAGGAAAAGGCTGAGGCTCTAGAGGACCTGGTTGGCTTCAAAGAACTCTTCCAGACACCAGGTCACACT GAGGAATCAATGACTGATGACAAAATCACAGAAGTATCCTGTAAATCTCCACAGCCAGAGTCATTCAAA

[0495] ACCTCAAGAAGCTCCAAGCAAAGGCTCAAGATACCCCTGGTGAAAGTGGACATGAAAGAAGAGCCCCTA GCAGTCAGCAAGCTCACACGGACATCAGGGGAGACTACGCAAACACACACAGAGCCAACAGGAGATAGT

[0496] AAGAGCATCAAAGCGTTTAAGGAGTCTCCAAAGCAGATCCTGGACCCAGCAGCAAGTGTAACTGGTAGC AGGAGGCAGCTGAGAACTCGTAAGGAAAAGGCCCGTGCTCTAGAAGACCTGGTTGACTTCAAAGAGCTC TTCTCAGCACCAGGTCACACTGAAGAGTCAATGACTATTGACAAAAACACAAAAATTCCCTGCAAATCTC CCCCACCAGAACTAACAGACACTGCCACGAGCACAAAGAGATGCCCCAAGACACGTCCCAGGAAAGAAG

[0497] TAAAAGAGGAGCTCTCAGCAGTTGAGAGGCTCACGCAAACATCAGGGCAAAGCACACACACACACAAAG AACCAGCAAGCGGTGATGAGGGCATCAAAGTATTGAAGCAACGTGCAAAGAAGAAACCAAACCCAGTA GAAGAGGAACCCAGCAGGAGAAGGCCAAGAGCACCTAAGGAAAAGGCCCAACCCCTGGAAGACCTGGC

[0498] CGGCTTCACAGAGCTCTCTGAAACATCAGGTCACACTCAGGAATCACTGACTGCTGGCAAAGCCACTAAA ATACCCTGCGAATCTCCCCCACTAGAAGTGGTAGACACCACAGCAAGCACAAAGAGGCATCTCAGGACA CGTGTGCAGAAGGTACAAGTAAAAGAAGAGCCTTCAGCAGTCAAGTTCACACAAACATCAGGGGAAACC

[0499] ACGGATGCAGACAAAGAACCAGCAGGTGAAGATAAAGGCATCAAAGCATTGAAGGAATCTGCAAAACA GACACCGGCTCCAGCAGCAAGTGTAACTGGCAGCAGGAGACGGCCAAGAGCACCCAGGGAAAGTGCCCA

[0500] AGCCATAGAAGACCTAGCTGGCTTCAAAGACCCAGCAGCAGGTCACACTGAAGAATCAATGACTGATGA CAAAACCACTAAAATACCCTGCAAATCATCACCAGAACTAGAAGACACCGCAACAAGCTCAAAGAGACG

[0501] GCCCAGGACACGTGCCCAGAAAGTAGAAGTGAAGGAGGAGCTGTTAGCAGTTGGCAAGCTCACACAAAC CTCAGGGGAGACCACGCACACCGACAAAGAGCCGGTAGGTGAGGGCAAAGGCACGAAAGCATTTAAGC

[0502] AACCTGCAAAGCGGAAGCTGGACGCAGAAGATGTAATTGGCAGCAGGAGACAGCCAAGAGCACCTAAG GAAAAGGCCCAACCCCTGGAAGATCTGGCCAGCTTCCAAGAGCTCTCTCAAACACCAGGCCACACTGAG

[0503] GAACTGGCAAATGGTGCTGCTGATAGCTTTACAAGCGCTCCAAAGCAAACACCTGACAGTGGAAAACCTC

[0504] TAAAAATATCCAGAAGAGTTCTTCGGGCCCCTAAAGTAGAACCCGTGGGAGACGTGGTAAGCACCAGAG ACCCTGTAAAATCACAAAGCAAAAGCAACACTTCCCTGCCCCCACTGCCCTTCAAGAGGGGAGGTGGCA

[0505] AAGATGGAAGCGTCACGGGAACCAAGAGGCTGCGCTGCATGCCAGCACCAGAGGAAATTGTGGAGGAGC

[0506] TGCCAGCCAGCAAGAAGCAGAGGGTTGCTCCCAGGGCAAGAGGCAAATCATCCGAACCCGTGGTCATCA TGAAGAGAAGTTTGAGGACTTCTGCAAAAAGAATTGAACCTGCGGAAGAGCTGAACAGCAACGACATGA AAACCAACAAAGAGGAACACAAATTACAAGACTCGGTCCCTGAAAATAAGGGAATATCCCTGCGCTCCA

[0507] GACGCCAAAATAAGACTGAGGCAGAACAGCAAATAACTGAGGTCTTTGTATTAGCAGAAAGAATAGAAA TAAACAGAAATGAAAAGAAGCCCATGAAGACCTCCCCAGAGATGGACATTCAGAATCCAGATGATGGAG CCCGGAAACCCATACCTAGAGACAAAGTCACTGAGAACAAAAGGTGCTTGAGGTCTGCTAGACAGAATG

[0508] AGAGCTCCCAGCCTAAGGTGGCAGAGGAGAGCGGAGGGCAGAAGAGTGCGAAGGTTCTCATGCAGAATC AGAAAGGGAAAGGAGAAGCAGGAAATTCAGACTCCATGTGCCTGAGATCAAGAAAGACAAAAAGCCAG CCTGCAGCAAGCACTTTGGAGAGCAAATCTGTGCAGAGAGTAACGCGGAGTGTCAAGAGGTGTGCAGAA

[0509] AATCCAAAGAAGGCTGAGGACAATGTGTGTGTCAAGAAAATAAGAACCAGAAGTCATAGGGACAGTGAA GATATTTGA

[0510] SEQ ID NO: 17 > Protein sequence for human Ki-67 full-length:

[0511] MWPTRRL VTIKRSGVDGPHFPL SL STCLFGRGIECDIRIQLP VVSKQHCKIEIHEQE AILHNFS STNPTQ VNGS VI DEPVRLKHGDVITIIDRSFRYENESLQNGRKSTEFPRKIREQEPARRVSRSSFSSDPDEKAQDSKAYSKITEGKVS GNPQVHIKNVKEDSTADDSKDSVAQGTTNVHSSEHAGRNGRNAADPISGDFKEISSVKLVSRYGELKSVPTTQ CLDNSKKNESPFWKLYESVKKELDVKSQKENVLQYCRKSGLQTDYATEKESADGLQGETQLLVSRKSRPKSG GSGHAVAEPASPEQELDQNKGKGRDVESVQTPSKAVGASFPLYEPAKMKTPVQYSQQQNSPQKHKNKDLYT TGRRESVNLGKSEGFKAGDKTLTPRKLSTRNRTPAKVEDAADSATKPENLSSKTRGSIPTDVEVLPTETEIHNE PFLTLWLTQVERKIQKDSLSKPEKLGTTAGQMCSGLPGLSSVDINNFGDSINESEGIPLKRRRVSFGGHLRPELF DENLPPNTPLKRGEAPTKRKSLVMHTPPVLKKIIKEQPQPSGKQESGSEIHVEVKAQSLVISPPAPSPRKTPVAS DQRRRSCKTAPASSSKSQTEVPKRGGRKSGNLPSKRVSISRSQHDILQMICSKRRSGASEANLIVAKSWADVVK LGAKQTQTKVIKHGPQRSMNKRQRRPATPKKPVGEVHSQFSTGHANSPCTIIIGKAHTEKVHVPARPYRVLNN FISNQKMDFKEDLSGIAEMFKTPVKEQPQLTSTCHIAISNSENLLGKQFQGTDSGEEPLLPTSESFGGNVFFSAQ NAAKQPSDKCSASPPLRRQCIRENGNVAKTPRNTYKMTSLETKTSDTETEPSKTVSTANRSGRSTEFRNIQKLP VESKSEETNTEIVECILKRGQKATLLQQRREGEMKEIERPFETYKENIELKENDEKMKAMKRSRTWGQKCAPM SDLTDLKSLPDTELMKDTARGQNLLQTQDHAKAPKSEKGKITKMPCQSLQPEPINTPTHTKQQLKASLGKVGV KEELLAVGKFTRTSGETTHTHREPAGDGKSIRTFKESPKQILDPAARVTGMKKWPRTPKEEAQSLEDLAGFKE LFQTPGPSEESMTDEKTTKIACKSPPPESVDTPTSTKQWPKRSLRKADVEEEFLALRKLTPSAGKAMLTPKPAG GDEKDIKAFMGTPVQKLDLAGTLPGSKRQLQTPKEKAQALEDLAGFKELFQTPGHTEELVAAGKTTKIPCDSP QSDPVDTPTSTKQRPKRSIRKADVEGELLACRNLMPSAGKAMHTPKPSVGEEKDIIIFVGTPVQKLDLTENLTG SKRRPQTPKEEAQALEDLTGFKELFQTPGHTEEAVAAGKTTKMPCESSPPESADTPTSTRRQPKTPLEKRDVQK ELSALKKLTQTSGETTHTDKVPGGEDKSINAFRETAKQKLDPAASVTGSKRHPKTKEKAQPLEDLAGLKELFQ TPVCTDKPTTHEKTTKIACRSQPDPVDTPTSSKPQSKRSLRKVDVEEEFFALRKRTPSAGKAMHTPKPAVSGEK NIYAFMGTPVQKLDLTENLTGSKRRLQTPKEKAQALEDLAGFKELFQTRGHTEESMTNDKTAKVACKSSQPD PDKNPASSKRRLKTSLGKVGVKEELLAVGKLTQTSGETTHTHTEPTGDGKSMKAFMESPKQILDSAASLTGSK RQLRTPKGKSEVPEDLAGFIELFQTPSHTKESMTNEKTTKVSYRASQPDLVDTPTSSKPQPKRSLRKADTEEEFL AFRKQTPSAGKAMHTPKPAVGEEKDINTFLGTPVQKLDQPGNLPGSNRRLQTRKEKAQALEELTGFRELFQTP CTDNPTTDEKTTKKILCKSPQSDPADTPTNTKQRPKRSLKKADVEEEFLAFRKLTPSAGKAMHTPKAAVGEEK DINTFVGTPVEKLDLLGNLPGSKRRPQTPKEKAKALEDLAGFKELFQTPGHTEESMTDDKITEVSCKSPQPDPV KTPTSSKQRLKISLGKVGVKEEVLPVGKLTQTSGKTTQTHRETAGDGKSIKAFKESAKQMLDPANYGTGMER WPRTPKEEAQSLEDLAGFKELFQTPDHTEESTTDDKTTKIACKSPPPESMDTPTSTRRRPKTPLGKRDIVEELSA LKQLTQTTHTDKVPGDEDKGINVFRETAKQKLDPAASVTGSKRQPRTPKGKAQPLEDLAGLKELFQTPICTDK PTTHEKTTKIACRSPQPDPVGTPTIFKPQSKRSLRKADVEEESLALRKRTPSVGKAMDTPKPAGGDEKDMKAF MGTPVQKLDLPGNLPGSKRWPQTPKEKAQALEDLAGFKELFQTPGTDKPTTDEKTTKIACKSPQPDPVDTPAS TKQRPKRNLRKADVEEEFLALRKRTPSAGKAMDTPKPAVSDEKNINTFVETPVQKLDLLGNLPGSKRQPQTPK EKAEALEDLVGFKELFQTPGHTEESMTDDKITEVSCKSPQPESFKTSRSSKQRLKIPLVKVDMKEEPLAVSKLT RTSGETTQTHIEPTGDSKSIKAFKESPKQILDPAASVTGSRRQLRTRKEKARALEDLVDFKELFSAPGHTEESM

[0512] TIDKNTKIPCKSPPPELTDTATSTKRCPKTRPRKEVKEELSAVERLTQTSGQSTHTHKEPASGDEGIKVLKQRAK KKPNPVEEEPSRRRPRAPKEKAQPLEDLAGFTELSETSGHTQESLTAGKATKIPCESPPLEVVDTTASTKRHLRT RVQKVQVKEEPSAVKFTQTSGETTDADKEPAGEDKGIKALKESAKQTPAPAASVTGSRRRPRAPRESAQAIED LAGFKDPAAGHTEESMTDDKTTKIPCKSSPELEDTATSSKRRPRTRAQKVEVKEELLAVGKLTQTSGETTHTD KEPVGEGKGTKAFKQPAKRKLDAEDVIGSRRQPRAPKEKAQPLEDLASFQELSQTPGHTEELANGAADSFTSA PKQTPDSGKPLKISRRVLRAPKVEPVGDVVSTRDPVKSQSKSNTSLPPLPFKRGGGKDGSVTGTKRLRCMPAPE EIVEELPASKKQRVAPRARGKSSEPVVIMKRSLRTSAKRIEPAEELNSNDMKTNKEEHKLQDSVPENKGISLRS RRQNKTEAEQQIIEVFVLAERIEINRNEKKPMKTSPEMDIQNPDDGARKPIPRDKVTENKRCLRSARQNESSQP KVAEESGGQKSAKVLMQNQKGKGEAGNSDSMCLRSRKTKSQPAASTLESKSVQRVTRSVKRCAENPKKAED NVCVKKIRTRSHRDSEDI

[0513] SEQ ID NO: 18 > DNA sequence for human Ki -67 DNA binding domain:

[0514] CACACTGAGGAACTGGCAAATGGTGCTGCTGATAGCTTTACAAGCGCTCCAAAGCAAACACCTGACAGTG GAAAACCTCTAAAAATATCCAGAAGAGTTCTTCGGGCCCCTAAAGTAGAACCCGTGGGAGACGTGGTAA GCACCAGAGACCCTGTAAAATCACAAAGCAAAAGCAACACTTCCCTGCCCCCACTGCCCTTCAAGAGGG GAGGTGGCAAAGATGGAAGCGTCACGGGAACCAAGAGGCTGCGCTGCATGCCAGCACCAGAGGAAATTG TGGAGGAGCTGCCAGCCAGCAAGAAGCAGAGGGTTGCTCCCAGGGCAAGAGGCAAATCATCCGAACCCG

[0515] TGGTCATCATGAAGAGAAGTTTGAGGACTTCTGCAAAAAGAATTGAACCTGCGGAAGAGCTGAACAGCA

[0516] ACGACATGAAAACCAACAAAGAGGAACACAAATTACAAGACTCGGTCCCTGAAAATAAGGGAATATCCC

[0517] TGCGCTCCAGACGCCAAAATAAGACTGAGGCAGAACAGCAAATAACTGAGGTCTTTGTATTAGCAGAAA

[0518] GAATAGAAATAAACAGAAATGAAAAGAAGCCCATGAAGACCTCCCCAGAGATGGACATTCAGAATCCAG

[0519] ATGATGGAGCCCGGAAACCCATACCTAGAGACAAAGTCACTGAGAACAAAAGGTGCTTGAGGTCTGCTA

[0520] GACAGAATGAGAGCTCCCAGCCTAAGGTGGCAGAGGAGAGCGGAGGGCAGAAGAGTGCGAAGGTTCTC

[0521] ATGCAGAATCAGAAAGGGAAAGGAGAAGCAGGAAATTCAGACTCCATGTGCCTGAGATCAAGAAAGAC

[0522] AAAAAGCCAGCCTGCAGCAAGCACTTTGGAGAGCAAATCTGTGCAGAGAGTAACGCGGAGTGTCAAGAG

[0523] GTGTGCAGAAAATCCAAAGAAGGCTGAGGACAATGTGTGTGTCAAGAAAATAAGAACCAGAAGTCATAG GGACAGTGAAGATATT

[0524] SEQ ID NO: 19 > DNA sequence for human Securin full length:

[0525] ATGGCTACTCTGATCTATGTTGATAAGGAAAATGGAGAACCAGGCACCCGTGTGGTTGCTAAGGATGGGC

[0526] TGAAGCTGGGGTCTGGACCTTCAATCAAAGCCTTAGATGGGAGATCTCAAGTTTCAACACCACGTTTTGG

[0527] CAAAACGTTCGATGCCCCACCAGCCTTACCTAAAGCTACTAGAAAGGCTTTGGGAACTGTCAACAGAGCT

[0528] ACAGAAAAGTCTGTAAAGACCAAGGGACCCCTCAAACAAAAACAGCCAAGCTTTTCTGCCAAAAAGATG

[0529] ACTGAGAAGACTGTTAAAGCAAAAAGCTCTGTTCCTGCCTCAGATGATGCCTATCCAGAAATAGAAAAAT

[0530] TCTTTCCCTTCAATCCTCTAGACTTTGAGAGTTTTGACCTGCCTGAAGAGCACCAGATTGCGCACCTCCCC

[0531] TTGAGTGGAGTGCCTCTCATGATCCTTGACGAGGAGAGAGAGCTTGAAAAGCTGTTTCAGCTGGGCCCCC

[0532] CTTCACCTGTGAAGATGCCCTCTCCACCATGGGAATCCAATCTGTTGCAGTCTCCTTCAAGCATTCTGTCG

[0533] ACCCTGGATGTTGAATTGCCACCTGTTTGCTGTGACATAGATATTTAAA

[0534] SEQ ID NO: 20 > Protein sequence for human Securin full length:

[0535] MATLIYVDKENGEPGTRVVAKDGLKLGSGPSIKALDGRSQVSTPRFGKTFDAPPALPKATRKALGTVNRATEK

[0536] SVKTKGPLKQKQPSFSAKKMTEKTVKAKSSVPASDDAYPEIEKFFPFNPLDFESFDLPEEHQIAHLPLSGVPLMI

[0537] LDEERELEKLFQLGPPSPVKMPSPPWESNLLQSPSSILSTLDVELPPVCCDIDI

[0538] SEQ ID NO: 21 > DNA sequence for human Securin separase-binding domain:

[0539] AGCTCTGTTCCTGCCTCAGATGATGCCTATCCAGAAATAGAAAAATTCTTTCCCTTCAATCCTCTAGACTT

[0540] TGAGAGTTTTGACCTGCCTGAAGAGCACCAGATTGCGCACCTCCCCTTGAGTGGAGTGCCTCTC

[0541] SEQ ID NO: 22 > DNA sequence for human Meikin Full Length:

[0542] ATGTGGCCGCTACGGGTCTATACCCGCAAAAAGCGGGAGGGTCAGAGGCTCAATCTCACCCCGACGCCA

[0543] GACCTAGGCTCTCCAGCGAAGGCCGAGGCCCCGCCAGGTTCGAAGAGAAAAGGCAAAGTGCACGGCTTG

[0544] TCGAAGATTGCAGAGAAAGCAGAGCGGAGCAGGCAGGGAGGTAGCGGCTCTGGGCCGTTCAGCCCTCGC

[0545] TTAGGAGTTACAGGAGAGAAAAGCCTGCAAGAAAATAGGTCTAGTGAAGACACCCAGGATGAGAAGATT

[0546] GCATCGTTGCGTGAATCAGTTACTGATGACCTCCAGGTTGATAGTAGTTCATCAAATAGTGAACTAGTATC

[0547] AGGATTAAGTTTGCACCATGGTATGGCCAGTTCTCTTCTGAGCTATTCAGTCACAGACTCTTATGCAGAAT

[0548] ACAAGAGTTTTGAAGAGAGCTTTCCATCACCTGAACTGTTCAGAAAATCAGATTATTTAGACTGGGAGTG

[0549] TCCCAACTTGGAAGAACACATGCAGTGGAAGAATTCTACTCTTCTGGATACCAGTAAAGCAGTAGCGATA

[0550] GAGAAGGCACCACAGTTTTCAAATGTCTCAGCAATTTTCAGTACCTCTTCAGAAGACTATCAGAAATGCC

[0551] ATAGAAAAACAGTGATGACAGTAGCAGATCAAAATGTTTCTCCAAAAGCAAAGTGTGCTTCAAATTCAG

[0552] AATCAGATAATGCAGCTTGTGAGATTTTACTTGCTGAGAAAACTTGCCCTTCAACCCCTGAAAAAACAAA

[0553] GAAAAAAAAAACAAATTCCAGTACTCCTGGTAAGAAAAACAGAGGCCTTTTAACAAGTACTCCATCTTCA GAGACAGCTGGTTTTGTGATTGATTTGTCCTCAGTGCAAAAAGCATCTTTTGAAGAACTATTTCCAAATGT CAGCAATTATGTTAATTCAAATGAAATTGTTCCTGTGTCAAGTTTGCAGGAAAATTCTTCAAATGAGTTTC CTGCAAATGCATCAGAAATATGTTGTATTATTAGAACATCACCAGGAACTAGACAAGTGAAAAATAAAG GTGTTATTGTAAAGAAGAAGAAATATTCTCTTCCTAAGGATACCCCTCAAGATATCATAATAAAAATGGC ATGA

[0554] SEQ ID NO: 23 > Protein sequence for human Meikin Full Length:

[0555] MWPLRVYTRKKREGQRLNLTPTPDLGSPAKAEAPPGSKRKGKVHGLSKIAEKAERSRQGGSGSGPFSPRLGVT GEKSLQENRSSEDTQDEKI ASLRES VTDDLQ VD S S S SNSEL VSGL SLHHGMAS SLL S YS VTD SYAEYKSFEESFP SPELFRKSDYLDWECPNLEEHMQWKNSTLLDTSKAVAIEKAPQFSNVSAIFSTSSEDYQKCHRKTVMTVADQ NVSPKAKCASNSESDNAACEILLAEKTCPSTPEKTKKKKTNSSTPGKKNRGLLTSTPSSETAGFVIDLSSVQKAS FEELFPNVSNYVNSNEIVPVSSLQENSSNEFPANASEICCIIRTSPGTRQVKNKGVIVKKKKYSLPKDTPQDIIIKM A

[0556] SEQ ID NO: 24 > DNA sequence for human Meikin separase-cleavage target:

[0557] GCCAGTTCTCTTCTGAGCTATTCAGTCACAGACTCTTATGCAGAATACAAGAGTTTTGAAGAGAGCTTTCC ATCACCTGAACTGTTCAGAAAATCAGATTATTTAGACTGGGAGTGTCCCAACTTGGAAGAACACATGCAG TGGAAGAATTCTACTCTTCTGGATACCAGTAAAGCAGTAGCG

[0558] SEQ ID NO: 25 > DNA sequence for human Rec8 Full Length:

[0559] ATGTTCTACTATCCCAACGTGCTTCAGCGCCACACCGGCTGCTTTGCCACCATCTGGCTGGCGGCGACTCG CGGCAGCCGGTTGGTGAAGCGCGAATACCTGAGGGTGAATGTGGTGAAAACCTGCGAGGAAATCCTCAA TTACGTGCTGGTACGAGTGCAACCCCCGCAGCCCGGCCTGCCGCGGCCCCGCTTCTCCCTCTATCTCTCAG CCCAACTTCAGATCGGTGTGATCCGCGTCTATTCTCAACAATGCCAGTACCTCGTGGAGGACATCCAGCA CATCTTGGAGCGCCTCCACCGTGCCCAGCTGCAGATCCGAATAGATATGGAGACTGAGCTACCCAGCCTG CTGCTTCCTAACCACCTGGCCATGATGGAGACCCTAGAAGATGCTCCAGATCCCTTTTTTGGGATGATGTC

[0560] TGTGGATCCCAGACTTCCTAGTCCTTTCGATATCCCTCAGATTCGACACCTCTTAGAGGCTGCAATCCCAG AGAGAGTTGAAGAGATCCCTCCTGAAGTTCCTACAGAGCCCAGGGAGCCAGAGAGGATTCCGGTCACTG TGCTGCCACCTGAGGCCATCACGATCCTGGAGGCAGAGCCCATACGGATGCTGGAGATTGAGGGTGAAC GGGAGCTCCCAGAGGTCAGCCGCCGAGAACTGGACCTGCTGATCGCAGAGGAAGAAGAAGCTATCTTGT TAGAAATCCCGCGGCTCCCACCTCCAGCTCCTGCAGAGGTGGAAGGAATAGGAGAGGCACTGGGTCCTG AGGAGCTGAGGCTGACAGGCTGGGAACCTGGGGCCCTACTCATGGAGGTGACCCCCCCGGAGGAGCTGC

[0561] GTCTGCCAGCCCCACCCAGCCCAGAGAGGAGGCCCCCAGTCCCCCCACCTCCTCGCCGCCGCCGTCGTCG CCGGTTACTGTTCTGGGACAAGGAGACTCAGATCTCCCCGGAGAAATTCCAGGAACAACTGCAAACCAG AGCCCACTGCTGGGAATGTCCTATGGTGCAGCCGCCCGAGAGGACCATCAGAGGCCCTGCGGAGTTGTTC

[0562] AGAACCCCAACTCTCTCTGGCTGGCTACCCCCTGAACTACTGGGTCTCTGGACCCATTGTGCCCAGCCACC CCCAAAAGCCCTCAGGCGAGAGCTGCCTGAGGAGGCAGCCGCTGAGGAGGAAAGGAGAAAGATTGAAG TTCCAAGTGAGATTGAGGTCCCGAGGGAGGCCCTGGAGCCCAGTGTTCCCCTTATGGTGTCTTTAGAGAT CTCCCTAGAGGCAGCTGAAGAGGAGAAGTCCCGCATCAGCCTCATCCCACCAGAAGAACGGTGGGCCTG GCCTGAGGTGGAGGCGCCAGAAGCTCCTGCATTGCCCGTGGTGCCTGAACTCCCTGAGGTGCCCATGGAG ATGCCTTTGGTGCTGCCCCCAGAGCTCGAGCTGCTCTCACTGGAAGCAGTGCACAGGGCAGTGGCACTGG

[0563] AGCTGCAGGCTAACAGGGAGCCCGACTTCAGCAGCCTGGTGTCACCTCTCAGCCCCCGCAGGATGGCTGC CCGGGTCTTCTACCTGCTCCTGGTGCTCTCAGCGCAACAGATTCTTCACGTGAAACAAGAAAAGCCATAT GGTCGCCTCCTGATCCAGCCGGGGCCCAGATTCCACTGA SEQ ID NO: 26 > Protein sequence for human Rec8 Full Length:

[0564] MFYYPNVLQRHTGCFATIWLAATRGSRLVKREYLRVNVVKTCEEILNYVLVRVQPPQPGLPRPRFSLYLSAQL

[0565] QIGVIRVYSQQCQYLVEDIQHILERLHRAQLQIRIDMETELPSLLLPNHLAMMETLEDAPDPFFGMMSVDPRLP

[0566] SPFDIPQIRHLLEAAIPERVEEIPPEVPTEPREPERIPVTVLPPEAITILEAEPIRMLEIEGERELPEVSRRELDLLIAE

[0567] EEEAILLEIPRLPPPAPAEVEGIGEALGPEELRLTGWEPGALLMEVTPPEELRLPAPPSPERRPPVPPPPRRRRRRR

[0568] LLFWDKETQISPEKFQEQLQTRAHCWECPMVQPPERTIRGPAELFRTPILSGWLPPELLGLWTHCAQPPPKALR

[0569] RELPEEAAAEEERRKIEVPSEIEVPREALEPSVPLMVSLEISLEAAEEEKSRISLIPPEERWAWPEVEAPEAPALPV

[0570] VPELPE VPMEMPL VLPPELELL SLE AVHRAVALELQ ANREPDFS SL VSPL SPRRMAARVFYLLL VL S AQQILH V KQEKPYGRLLIQPGPRFH

[0571] SEQ ID NO: 27 > DNA sequence for human Rec8 separase-cleavage target:

[0572] AGAACCCCAACTCTCTCTGGCTGGCTACCCCCTGAACTACTGGGTCTCTGGACCCATTGTGCCCAGCCACC

[0573] CCCAAAAGCCCTCAGGCGAGAGCTGCCTGAGGAGGCAGCCGCTGAGGAGGAAAGGAGAAAGATTGAAG

[0574] TTCCAAGTGAGATTGAGGTCCCGAGGGAGGCCCTGGAGCCCAGTGTTCCCCTTATGGTGTCTTTAGAGAT

[0575] CTCCCTAGAGGCAGCTGAAGAGGAGAAGTCCCGCATCAGCCTCATCCCACCAGAAGAACGGTGGGCCTG

[0576] GCCTGAGGTGGAGGCGCCAGAAGCTCCTGCATTGCCCGTGGTGCCTGAACTCCCTGAGGTGCCCATGGAG

[0577] ATGCCTTTGGTGCTGCCCCCAGAGCTCGAGCTGCTCTCACTGGAAGCAGTGCACAGGGCAGTGGCA

[0578] SEQ ID NO: 28 > DNA sequence for human Shugoshin 1 Full Length:

[0579] ATGGCCAAGGAAAGATGCCTGAAAAAGTCCTTTCAAGATAGTCTTGAAGACATAAAGAAGCGAATGAAA

[0580] GAGAAAAGGAATAAAAACTTGGCAGAGATTGGCAAACGCAGGTCTTTTATAGCTGCACCATGCCAAATA

[0581] ATCACCAACACTTCTACACTGCTGAAAAATTACCAAGACAACAACAAAATGTTAGTTTTAGCTTTGGAAA

[0582] ATGAAAAATCCAAAGTGAAAGAAGCCCAAGATATCATCCTACAGCTGAGAAAAGAATGTTACTATCTCA

[0583] CATGTCAGCTATATGCATTGAAAGGAAAACTTACATCACAACAAACAGTAGAACCTGCTCAGAACCAGG

[0584] AAATATGTTCCTCTGGAATGGACCCCAATAGTGATGACAGCTCCAGAAATTTATTTGTGAAGGATTTACC

[0585] GCAAATTCCTCTTGAAGAAACTGAACTTCCAGGACAAGGAGAATCATTTCAAATAGAAGATCAGATACCT

[0586] ACTATTCCTCAAGACACACTGGGAGTTGATTTTGATTCAGGTGAAGCTAAGTCTACTGATAATGTCTTACC

[0587] TAGAACTGTATCTGTTCGTAGCAGTTTAAAGAAACATTGTAACAGTATATGTCAGTTTGATAGCTTGGATG

[0588] ATTTTGAAACCAGTCATTTGGCAGGGAAGTCTTTTGAATTCGAAAGAGTTGGATTTTTAGACCCACTAGTA

[0589] AACATGCACATACCTGAAAATGTACAACACAATGCTTGTCAATGGAGCAAGGACCAAGTTAACTTATCAC

[0590] CAAAGCTGATTCAGCCAGGAACGTTTACTAAAACAAAAGAAGACATTTTAGAATCTAAATCTGAACAAA

[0591] CTAAAAGTAAGCAAAGAGATACACAAGAAAGAAAAAGAGAAGAGAAAAGAAAAGCTAACAGGAGAAA

[0592] ATCAAAACGTATGTCAAAATATAAAGAGAATAAAAGCGAAAATAAAAAAACTGTTCCCCAAAAAAAAAT

[0593] GCACAAATCTGTCAGTTCCAATGATGCTTACAATTTTAATTTGGAAGAGGGTGTTCATCTTACTCCTTTCC

[0594] GACAAAAAGTGAGCAATGACTCTAATAGAGAAGAAAACAACGAGTCTGAAGTGAGCCTCTGTGAATCAA

[0595] GTGGTTCAGGAGATGATTCCGATGACCTCTATTTGCCCACTTGCAAGTACATTCAGAATCCCACGAGCAA

[0596] TTCAGATAGACCAGTCACCAGGCCTCTAGCTAAAAGAGCACTGAAATACACAGATGAAAAAGAGACGGA

[0597] GGGTTCTAAGCCAACAAAAACTCCTACCACTACACCACCTGAAACTCAGCAGTCACCTCATCTTAGCCTG

[0598] AAGGATATCACCAATGTCTCCTTGTATCCTGTTGTGAAAATCAGAAGACTTTCTCTTTCTCCAAAAAAGAA

[0599] TAAAGCAAGCCCAGCAGTGGCTCTGCCTAAACGTAGGTGCACAGCCAGCGTGAACTATAAGGAGCCCAC

[0600] CCTCGCTTCGAAACTGAGAAGAGGGGACCCTTTTACAGATTTGTGTTTTTTGAATTCTCCTATTTTCAAGC

[0601] AGAAAAAGGATTTGAGACGTTCTAAAAAAAGAGCCCTGGAGGTATCACCTGCCAAAGAAGCAATTTTTA

[0602] TTTTATATTATGTTCGAGAATTTGTTTCGAGATTCCCAGACTGTAGGAAATGTAAACTTGAAACCCACATC

[0603] TGCTTGAGGTAA SEQ ID NO: 29> Protein sequence for human Shugoshin 1 Full Length:

[0604] MAKERCLKKSFQDSLEDIKKRMKEKRNKNLAEIGKRRSFIAAPCQIITNTSTLLKNYQDNNKMLVLALENEKS

[0605] KVKEAQDIILQLRKECYYLTCQLYALKGKLTSQQTVEPAQNQEICSSGMDPNSDDSSRNLFVKDLPQIPLEETE

[0606] LPGQGESFQIEDQIPTIPQDTLGVDFDSGEAKSTDNVLPRTVSVRSSLKKHCNSICQFDSLDDFETSHLAGKSFEF

[0607] ERVGFLDPLVNMHIPENVQHNACQWSKDQVNLSPKLIQPGTFTKTKEDILESKSEQTKSKQRDTQERKREEKR

[0608] KANRRKSKRMSKYKENKSENKKTVPQKKMHKSVSSNDAYNFNLEEGVHLTPFRQKVSNDSNREENNESEVS

[0609] LCESSGSGDDSDDLYLPTCKYIQNPTSNSDRPVTRPLAKRALKYTDEKETEGSKPTKTPTTTPPETQQSPHLSLK DITNVSLYP VVKJRRL SL SPKKNKASP AVALPKRRCT AS VNYKEPTL ASKLRRGDPFIDLCFLNSPIFKQKKDLR RSKKRALEVSPAKEAIFILYYVREFVSRFPDCRKCKLETHICLR

[0610] SEQ ID NO: 30> DNA sequence for human Shugoshin 1 PP2A binding site:

[0611] TCTACACTGCTGAAAAATTACCAAGACAACAACAAAATGTTAGTTTTAGCTTTGGAAAATGAAAAATCCA

[0612] AAGTGAAAGAAGCCCAAGATATCATCCTACAGCTGAGAAAAGAATGTTACTATCTCACATGTCAGCTA

[0613] SEQ ID NO: 31 > DNA sequence for human Shugoshin 2 Full Length:

[0614] ATGGAGTGCCCAGTGATGGAAACTGGCTCACTTTTTACCTCAGGAATTAAGAGACATTTGAAAGACAAAA

[0615] GAATTTCAAAGACTACTAAGTTGAATGTTTCTCTTGCTTCAAAAATAAAAACAAAAATACTAAATAATTC

[0616] TTCTATTTTCAAAATATCTTTAAAGCACAACAACAGGGCATTAGCTCAGGCTCTTAGTAGAGAAAAAGAG

[0617] AATTCTCGAAGAATTACAACTGAAAAGATGCTATTGCAAAAAGAAGTAGAGAAACTGAATTTTGAGAAC

[0618] ACATTTCTTCGCCTAAAGCTAAATAACTTGAATAAGAAGCTTATAGACATAGAAGCTCTCATGAACAATA

[0619] ACTTGATAACTGCAATTGAAATGAGCAGTCTTTCTGAGTTCCATCAGAGTTCCTTTCTACTGTCAGCTAGC

[0620] AAGAAGAAACGAATTAGTAAACAGTGCAAGTTGATGCGTCTTCCATTTGCAAGGGTTCCATTAACTTCAA

[0621] ATGATGATGAAGATGAAGATAAAGAGAAAATGCAGTGTGACAACAATATTAAATCAAAGACATTACCTG

[0622] ATATTCCCTCTTCAGGATCAACAACACAACCTTTATCAACTCAGGATAATTCGGAAGTGTTATTTCTTAAA

[0623] GAAAATAATCAAAATGTATATGGTTTAGATGATTCAGAACATATTTCTTCTATAGTTGATGTACCTCCCAG

[0624] AGAAAGCCATTCCCACTCAGACCAAAGTTCTAAGACTTCTCTAATGAGTGAGATGAGAAACGCCCAGTCT

[0625] ATTGGCCGCAGATGGGAGAAACCATCTCCTAGTAATGTGACTGAAAGGAAGAAGCGTGGGTCATCTTGG

[0626] GAATCAAATAATCTTTCTGCAGACACTCCCTGTGCAACAGTTTTAGATAAACAACACATTTCAAGTCCAG

[0627] AATTAAATTGCAATAATGAGATAAATGGTCATACTAATGAAACAAATACTGAAATGCAAAGAAATAAAC

[0628] AGGATCTTCCTGGCTTATCTTCTGAGTCTGCCAGAGAACCTAATGCAGAGTGCATGAATCAAATTGAGGA

[0629] TAATGATGACTTTCAATTGCAGAAAACTGTGTATGATGCTGACATGGATTTAACTGCTAGTGAAGTCAGC

[0630] AAAATTGTCACAGTCTCAACAGGCATTAAAAAGAAAAGTAATAAAAAAACAAATGAACATGGAATGAAA

[0631] ACTTTCAGAAAAGTGAAAGATTCCAGCTCTGAAAAAAAGAGAGAAAGATCAAAGAGACAGTTTAAAAAT

[0632] AGTTCAGATGTCGATATTGGGGAAAAGATTGAAAACAGGACAGAAAGATCTGATGTCCTGGATGGCAAA

[0633] AGGGGTGCAGAAGATCCCGGTTTTATTTTCAATAATGAACAGCTGGCTCAGATGAATGAACAGCTGGCTC

[0634] AGGTGAATGAACTAAAGAAAATGACCCTTCAAACTGGCTTTGAACAAGGTGACAGAGAAAATGTACTGT

[0635] GTAATAAAAAGGAGAAAAGAATAACAAATGAGCAAGAGGAAACATACTCTTTATCCCAAAGTTCAGGTA

[0636] AATTTCACCAGGAGAGTAAATTTGATAAGGGTCAGAATTCCCTAACTTGTAATAAAAGTAAAGCTTCTAG

[0637] ACAGACATTTGTGATTCACAAATTAGAAAAAGATAACTTACTCCCAAACCAAAAGGATAAAGTAACCATT

[0638] TATGAAAACCTAGACGTCACAAATGAATTTCACACAGCCAATCTTTCCACCAAAGATAATGGAAATTTAT

[0639] GTGATTATGGGACCCACAATATATTGGATTTGAAAAAGTATGTCACTGATATTCAACCCTCAGAGCAAAA

[0640] TGAATCAAACATTAATAAGCTTAGAAAGAAAGTAAACCGGAAGACAGAAATAATTTCTGGAATGAACCA

[0641] CATGTATGAGGATAATGATAAAGATGTGGTGCATGGCCTAAAAAAAGGTAATTTTTTTTTCAAAACCCAA

[0642] GAGGATAAAGAACCTATCTCTGAAAACATAGAAGTTTCCAAAGAGCTTCAAATCCCAGCTCTTTCTACTA

[0643] GAGATAATGAAAATCAATGTGACTATAGGACCCAGAATGTGTTGGGTTTGCAAAAGCAGATCACCAATAT GTACCCCGTTCAGCAAAATGAATCAAAAGTTAATAAGAAGCTTAGGCAGAAAGTAAATCGGAAGACAGA AATAATTTCTGAAGTGAATCATTTAGATAATGACAAAAGTATAGAATACACAGTTAAAAGTCACTCACTC TTTTTAACGCAAAAAGATAAGGAAATCATCCCTGGAAACCTAGAAGACCCAAGTGAGTTTGAAACACCTG CTCTTTCTACCAAAGATAGTGGAAACCTGTATGATTCTGAGATTCAAAATGTTTTGGGGGTGAAACATGG CCATGATATGCAACCTGCTTGTCAAAATGATTCAAAAATAGGTAAGAAGCCTAGACTAAATGTATGTCAA AAGTCAGAAATAATTCCTGAAACCAACCAAATATATGAGAATGATAACAAAGGTGTACATGACCTAGAA AAAGATAACTTCTTCTCTCTAACCCCAAAGGATAAAGAAACAATTTCTGAAAATCTACAAGTCACAAATG AATTTCAAACAGTTGATCTTCTCATCAAAGATAATGGAAATTTATGTGATTATGACACCCAGAATATATTG GAGTTGAAAAAGTATGTTACTGATAGGAAATCTGCTGAGCAAAATGAATCAAAAATAAATAAGCTCAGG AATAAAGTGAATTGGAAGACAGAAATAATTTCTGAAATGAACCAGATATATGAGGATAATGATAAAGAT GCACATGTCCAAGAAAGCTATACAAAAGATCTTGATTTTAAAGTAAATAAATCTAAACAAAAACTTGAAT GCCAAGACATTATCAATAAACACTATATGGAAGTCAACAGTAATGAAAAGGAAAGTTGTGATCAAATTTT AGATTCCTACAAAGTAGTTAAAAAACGTAAGAAAGAATCATCATGCAAGGCAAAGAACATTTTGACAAA AGCTAAGAACAAACTTGCTTCACAGTTAACAGAATCTTCACAGACATCTATCTCCTTAGAATCTGATTTAA AACATATTACTAGTGAAGCAGATTCTGATCCAGGAAACCCAGTTGAACTATGTAAGACTCAGAAGCAAA GCACTACCACTTTGAATAAAAAAGATCTCCCTTTTGTGGAAGAAATAAAAGAAGGAGAGTGTCAGGTTAA AAAGGTAAATAAAATGACATCTAAGTCAAAGAAAAGGAAGACCTCCATAGATCCTTCTCCAGAGAGCCA TGAAGTAATGGAAAGAATACTTGACAGCGTTCAGGGAAAGTCTACTGTATCTGAACAAGCTGATAAGGA AAACAATTTGGAGAATGAGAAAATGGTCAAAAATAAGCCAGACTTTTACACAAAGGCATTTAGATCTTTG TCTGAGATACATTCACCTAACATACAAGATTCTTCCTTTGACAGTGTTCGTGAAGGTTTAGTACCTTTGAG CGTTTCTTCTGGTAAAAATGTGATAATAAAAGAAAATTTTGCCTTGGAGTGCTCCCCAGCCTTTCAAGTAA GTGATGATGAGCATGAGAAGATGAACAAGATGAAATTTAAAGTCAACCGGAGAACCCAAAAATCAGGAA TAGGTGATAGACCATTACAGGACTTGTCAAATACCAGTTTTGTTTCAAATAACACTGCTGAATCTGAAAA TAAGTCAGAAGATCTATCTTCAGAACGGACAAGCAGAAGAAGAAGGTGTACTCCTTTCTATTTTAAAGAG CCAAGCCTCAGAGACAAGATGAGAAGATGA

[0644] SEQ ID NO: 32 > Protein sequence for human Shugoshin 2 Full Length:

[0645] MECPVMETGSLFTSGIKRHLKDKRISKTTKLNVSLASKIKTKILNNSSIFKISLKHNNRALAQALSREKENSRRIT TEKMLLQKE VEKLNFENTFLRLKLNNLNKKLIDIE ALMNNNLIT AIEMS SL SEFHQS SFLL S ASKKKRISKQCKL MRLPFARVPLTSNDDEDEDKEKMQCDNNIKSKTLPDIPSSGSTTQPLSTQDNSEVLFLKENNQNVYGLDDSEHI SSIVDVPPRESHSHSDQSSKTSLMSEMRNAQSIGRRWEKPSPSNVIERKKRGSSWESNNLSADTPCATVLDKQ HISSPELNCNNEINGHTNETNTEMQRNKQDLPGLSSESAREPNAECMNQIEDNDDFQLQKTVYDADMDLTASE VSKI VT VSTGIKKKSNKKTNEHGMKTFRKVKD S S SEKKRERSKRQFKNS SD VDIGEKIENRTERSD VLDGKRG AEDPGFIFNNEQLAQMNEQLAQVNELKKMTLQTGFEQGDRENVLCNKKEKRITNEQEETYSLSQSSGKFHQES KFDKGQNSLTCNKSKASRQTFVIHKLEKDNLLPNQKDKVTIYENLDVTNEFHTANLSTKDNGNLCDYGTHNIL DLKKYVTDIQPSEQNESNINKLRKKVNRKTEIISGMNHMYEDNDKDVVHGLKKGNFFFKTQEDKEPISENIEVS KELQIPALSTRDNENQCDYRTQNVLGLQKQITNMYPVQQNESKVNKKLRQKVNRKTEIISEVNHLDNDKSIEY TVKSHSLFLTQKDKEIIPGNLEDPSEFETPALSTKDSGNLYDSEIQNVLGVKHGHDMQPACQNDSKIGKKPRLN VCQKSEUPETNQIYENDNKGVHDLEKDNFFSLTPKDKETISENLQVTNEFQTVDLLIKDNGNLCDYDTQNILEL KKYVTDRKSAEQNESKINKLRNKVNWKTEIISEMNQIYEDNDKDAHVQESYTKDLDFKVNKSKQKLECQDII NKHYMEVNSNEKESCDQILDSYKVVKKRKKESSCKAKNILTKAKNKLASQLTESSQTSISLESDLKHITSEADS DPGNPVELCKTQKQSTTTLNKKDLPFVEEIKEGECQVKKVNKMTSKSKKRKTSIDPSPESHEVMERILDSVQG KST VSEQ ADKENNLENEKMVKNKPDFYTKAFRSL SEIHSPNIQD S SFD S VREGL VPL S VS SGKNVIIKENF ALEC SPAFQVSDDEHEKMNKMKFKVNRRTQKSGIGDRPLQDLSNTSFVSNNTAESENKSEDLSSERTSRRRRCTPFY FKEPSLRDKMRR SEQ ID NO: 33 > DNA sequence for human Shugoshin 2 PP2A binding site:

[0646] TCTTCTATTTTCAAAATATCTTTAAAGCACAACAACAGGGCATTAGCTCAGGCTCTTAGTAGAGAAAAAG

[0647] AGAATTCTCGAAGAATTACAACTGAAAAGATGCTATTGCAAAAAGAAGTAGAGAAACTGAATTTTGAGA ACACA

[0648] SEQ ID NO: 34 > DNA sequence for human Striatin3 full length:

[0649] ATGGACGAGCTTGCCGGAGGCGGTGGTGGCGGCCCGGGGATGGCGGCCCCTCCCCGGCAGCAGCAGGGA

[0650] CCTGGGGGGAACCTGGGCCTTTCGCCCGGGGGGAACGGAGCGGCGGGCGGCGGGGGTCCTCCGGCCTCC

[0651] GAGGGAGCGGGTCCCGCGGCAGGCCCCGAGCTGTCCCGGCCGCAGCAGTACACTATCCCGGGGATACTG

[0652] CACTACATCCAGCACGAGTGGGCTCGGTTCGAGATGGAGCGGGCGCACTGGGAGGTGGAACGGGCCGAA

[0653] CTGCAGGCCCGGATTGCATTTCTACAAGGCGAAAGAAAAGGTCAAGAGAACCTGAAGAAGGACTTAGTA

[0654] AGAAGAATAAAGATGTTAGAGTATGCATTAAAACAAGAAAGGGCAAAATATCACAAATTAAAATATGGC

[0655] ACGGAACTGAACCAAGGTGACTTGAAAATGCCAACCTTTGAGTCAGAAGAAACCAAAGACACAGAGGCT

[0656] CCCACAGCACCTCAGAATAGCCAGTTAACGTGGAAGCAAGGCAGACAGCTTTTAAGACAGTATCTTCAGG

[0657] AAGTAGGTTATACAGATACAATATTAGATGTACGGTCTCAGCGGGTAAGGTCATTACTTGGACTATCTAA

[0658] TTCAGAACCAAATGGATCAGTAGAAACAAAGAATTTAGAACAGATCCTGAATGGAGGTGAATCTCCTAA

[0659] GCAAAAGGGACAAGAAATAAAAAGGTCCTCTGGTGATGTTCTTGAGACGTTCAATTTCTTAGAAAATGCC

[0660] GATGACAGTGATGAAGATGAGGAAAATGACATGATCGAAGGCATCCCAGAAGGAAAAGACAAACATCG

[0661] GATGAATAAACATAAAATAGGTAATGAAGGTTTAGCTGCTGACCTAACTGACGATCCTGATACTGAGGAA

[0662] GCACTGAAAGAATTTGATTTTTTAGTGACTGCTGAAGATGGTGAAGGAGCTGGAGAAGCACGGAGTTCGG

[0663] GGGATGGCACAGAATGGGATAAAGATGACCTCTCCCCAACTGCTGAGGTTTGGGATGTAGACCAGGGAC

[0664] TAATAAGTAAACTGAAGGAACAGTACAAGAAGGAACGAAAGGGGAAGAAAGGGGTGAAGAGGGCCAAC

[0665] AGGACAAAACTCTACGACATGATAGCTGATCTGGGAGATGATGAGCTGCCCCACATCCCTTCAGGAATCA

[0666] TTAATCAGTCTAGGTCAGCCTCTACTAGAATGACTGATCATGAAGGTGCAAGAGCAGAGGAAGCTGAACC

[0667] AATAACGTTTCCATCTGGAGGAGGCAAGTCATTTATTATGGGTTCTGATGATGTTTTGTTAAGTGTACTGG

[0668] GCCTTGGAGACCTTGCAGACTTGACGGTAACAAATGATGCAGACTATAGTTATGATTTGCCTGCTAATAA

[0669] AGATGCCTTTCGAAAGACATGGAATCCCAAGTATACACTACGTAGCCATTTTGATGGAGTACGGGCATTA

[0670] GCTTTTCATCCTGTAGAACCTGTGCTGGTTACTGCTTCTGAGGACCATACCCTGAAACTTTGGAACCTGCA

[0671] AAAAACAGTTCCTGCCAAAAAGAGTGCCTCTTTAGATGTAGAGCCTATCTACACATTTAGGGCCCACATC

[0672] GGCCCTGTTCTGTCATTAGCTATTAGTTCTAATGGAGAACAGTGTTTTAGTGGTGGTATTGATGCAACCAT

[0673] CCAGTGGTGGAATATGCCGAGTCCCAGTGTAGATCCATATGATACATATGAGCCAAATGTTCTAGCTGGC

[0674] ACTTTAGTTGGTCATACAGATGCAGTTTGGGGTCTTGCTTATAGTGGCATAAAAAATCAATTACTGTCTTG

[0675] TTCAGCAGATGGCACTGTTAGGTTATGGAATCCACAAGAAAAATTGCCATGTATTTGCACTTACAATGGA

[0676] GATAAAAAGCATGGAATACCTACATCAGTTGACTTTATAGGCTGTGATCCAGCTCATATGGTAACCTCTTT

[0677] CAACACTGGTAGTGCAGTAATTTATGATTTAGAAACATCACAGTCATTGGTGATACTTTCATCACAGGTA

[0678] GATTCTGGTTTACAATCTAATAATCATATCAACAGAGTAGTAAGTCATCCCACACTTCCTGTTACAATAAC

[0679] TGCTCATGAAGATAGACACATCAAATTTTTTGACAATAAAACGGGTAAAATGATCCATTCTATGGTAGCT

[0680] CACTTGGATGCTGTTACAAGTCTAGCAGTAGATCCTAATGGAATCTATTTGATGTCTGGAAGCCATGACTG

[0681] TTCCATCAGATTATGGAATTTAGACAGCAAGACATGTGTGCAAGAAATAACAGCTCACAGAAAGAAATT

[0682] GGATGAATCAATTTATGATGTTGCTTTCCACTCGTCAAAAGCATATATAGCTAGTGCAGGAGCTGATGCTC TTGCCAAAGTATTTGTATGA

[0683] SEQ ID NO: 35 > Protein sequence for human Striatin3 full length: MDELAGGGGGGPGMAAPPRQQQGPGGNLGLSPGGNGAAGGGGPPASEGAGPAAGPELSRPQQYTIPGILHYI

[0684] QHEWARFEMERAHWEVERAELQARIAFLQGERKGQENLKKDLVRRIKMLEYALKQERAKYHKLKYGTELN

[0685] QGDLKMPTFESEETKDTEAPTAPQNSQLTWKQGRQLLRQYLQEVGYTDTILDVRSQRVRSLLGLSNSEPNGSV

[0686] ETKNLEQILNGGESPKQKGQEIKRSSGDVLETFNFLENADDSDEDEENDMIEGIPEGKDKHRMNKHKIGNEGL

[0687] AADLTDDPDTEEALKEFDFLVTAEDGEGAGEARSSGDGTEWDKDDLSPTAEVWDVDQGLISKLKEQYKKER

[0688] KGKKGVKRANRTKLYDMIADLGDDELPHIPSGIINQSRSASTRMTDHEGARAEEAEPITFPSGGGKSFIMGSDD

[0689] VLLSVLGLGDLADLTVTNDADYSYDLPANKDAFRKTWNPKYTLRSHFDGVRALAFHPVEPVLVTASEDHTLK

[0690] LWNLQKTVPAKKSASLDVEPIYTFRAHIGPVLSLAISSNGEQCFSGGIDATIQWWNMPSPSVDPYDTYEPNVLA

[0691] GTLVGHTDAVWGLAYSGIKNQLLSCSADGTVRLWNPQEKLPCICTYNGDKKHGIPTSVDFIGCDPAHMVTSF

[0692] NTGSAVIYDLETSQSLVILSSQVDSGLQSNNHINRVVSHPTLPVTITAHEDRHIKFFDNKTGKMIHSMVAHLDA

[0693] VTSLAVDPNGIYLMSGSHDCSIRLWNLDSKTCVQEITAHRKKLDESIYDVAFHSSKAYIASAGADALAKVFV

[0694] SEQ ID NO: 36> DNA sequence for human Striatin3 PP2A binding site:

[0695] TGGGAGGTGGAACGGGCCGAACTGCAGGCCCGGATTGCATTTCTACAAGGCGAAAGAAAAGGTCAAGAG

[0696] AACCTGAAGAAGGACTTAGTAAGAAGAATAAAGATGTTAGAGTAT

[0697] SEQ ID NO:37 > DNA sequence for human PP2A scaffold subunit A (PPP2A):

[0698] ATGGCGGCGGCCGACGGCGACGACTCGCTGTACCCCATCGCGGTGCTCATAGACGAACTCCGCAATGAG

[0699] GACGTTCAGCTTCGCCTCAACAGCATCAAGAAGCTGTCCACCATCGCCTTGGCCCTTGGGGTTGAAAGGA

[0700] CCCGAAGTGAGCTTCTGCCTTTCCTTACAGATACCATCTATGATGAAGATGAGGTCCTCCTGGCCCTGGCA

[0701] GAACAGCTGGGAACCTTCACTACCCTGGTGGGAGGCCCAGAGTACGTGCACTGCCTGCTGCCACCGCTGG

[0702] AGTCGCTGGCCACAGTGGAGGAGACAGTGGTGCGGGACAAGGCAGTGGAGTCCTTACGGGCCATCTCAC

[0703] ACGAGCACTCGCCCTCTGACCTGGAGGCGCACTTTGTGCCGCTAGTGAAGCGGCTGGCGGGCGGCGACTG

[0704] GTTCACCTCCCGCACCTCGGCCTGCGGCCTCTTCTCCGTCTGCTACCCCCGAGTGTCCAGTGCTGTGAAGG

[0705] CGGAACTTCGACAGTACTTCCGGAACCTGTGCTCAGATGACACCCCCATGGTGCGGCGGGCCGCAGCCTC

[0706] CAAGCTGGGGGAGTTTGCCAAGGTGCTGGAGCTGGACAACGTCAAGAGTGAGATCATCCCCATGTTCTCC

[0707] AACCTGGCCTCTGACGAGCAGGACTCGGTGCGGCTGCTGGCGGTGGAGGCGTGCGTGAACATCGCCCAG

[0708] CTTCTGCCCCAGGAGGATCTGGAGGCCCTGGTGATGCCCACTCTGCGCCAGGCCGCTGAAGACAAGTCCT

[0709] GGCGCGTCCGCTACATGGTGGCTGACAAGTTCACAGAGCTCCAGAAAGCAGTGGGGCCTGAGATCACCA

[0710] AGACAGACCTGGTCCCTGCCTTCCAGAACCTGATGAAAGACTGTGAGGCCGAGGTGAGGGCCGCAGCCT

[0711] CCCACAAGGTCAAAGAGTTCTGTGAAAACCTCTCAGCTGACTGTCGGGAGAATGTGATCATGTCCCAGAT

[0712] CTTGCCCTGCATCAAGGAGCTGGTGTCCGATGCCAACCAACATGTCAAGTCTGCCCTGGCCTCAGTCATC

[0713] ATGGGTCTCTCTCCCATCTTGGGCAAAGACAACACCATCGAGCACCTCTTGCCCCTCTTCCTGGCTCAGCT

[0714] GAAGGATGAGTGCCCTGAGGTACGGCTGAACATCATCTCTAACCTGGACTGTGTGAACGAGGTGATTGGC

[0715] ATCCGGCAGCTGTCCCAGTCCCTGCTCCCTGCCATTGTGGAGCTGGCTGAGGACGCCAAGTGGCGGGTGC

[0716] GGCTGGCCATCATTGAGTACATGCCCCTCCTGGCTGGACAGCTGGGAGTGGAGTTCTTTGATGAGAAACT

[0717] TAACTCCTTGTGCATGGCCTGGCTTGTGGATCATGTATATGCCATCCGCGAGGCAGCCACCAGCAACCTG

[0718] AAGAAGCTAGTGGAAAAGTTTGGGAAGGAGTGGGCCCATGCCACAATCATCCCCAAGGTCTTGGCCATG

[0719] TCCGGAGACCCCAACTACCTGCACCGCATGACTACGCTCTTCTGCATCAATGTGCTGTCTGAGGTCTGTGG

[0720] GCAGGACATCACCACCAAGCACATGCTACCCACGGTTCTGCGCATGGCTGGGGACCCGGTTGCCAATGTC

[0721] CGCTTCAATGTGGCCAAGTCTCTGCAGAAGATAGGGCCCATCCTGGACAACAGCACCTTGCAGAGTGAAG

[0722] TCAAGCCCATCCTAGAGAAGCTGACCCAGGACCAGGATGTGGACGTCAAATACTTTGCCCAGGAGGCTCT

[0723] GACTGTTCTGTCTCTCGCCTGA

[0724] SEQ ID NO:38 > DNA sequence for human PP2A Regulatory subunit B’ gamma (PPP2R5C): ATGCCGAATAAAAACAAGAAGGAGAAAGAATCACCAAAAGCAGGGAAGAGTGGAAAAAGTTCAAAAGA

[0725] AGGACAAGACACAGTAGAATCAGAGCAAATTTCCGTCAGGAAAAACAGCCTTGTTGCTGTCCCGTCTACA

[0726] GTATCTGCTAAAATAAAAGTACCAGTCTCTCAGCCCATAGTGAAGAAAGACAAACGGCAAAATTCTTCAA

[0727] GGTTTAGCGCAAGCAATAATAGAGAACTTCAAAAACTACCATCCTTAAAAGATGTTCCTCCTGCTGATCA

[0728] AGAGAAGCTTTTTATCCAGAAGTTACGTCAGTGTTGCGTCCTCTTTGACTTTGTTTCTGATCCACTAAGTG

[0729] ACCTAAAGTGGAAGGAAGTAAAACGAGCTGCTTTAAGTGAAATGGTAGAATATATCACCCATAATCGGA

[0730] ATGTGATCACAGAGCCTATTTACCCAGAAGTAGTCCATATGTTTGCAGTTAACATGTTTCGAACATTACCA

[0731] CCTTCCTCCAATCCTACGGGAGCGGAATTTGACCCGGAGGAAGATGAACCAACGTTAGAAGCAGCCTGGC

[0732] CTCATCTACAGCTTGTTTATGAATTTTTCTTAAGATTTTTAGAGTCTCCAGATTTCCAACCTAATATAGCGA

[0733] AGAAATATATTGATCAGAAGTTTGTATTGCAGCTTTTAGAGCTCTTTGACAGTGAAGATCCTCGGGAGAG

[0734] AGATTTTCTTAAAACCACCCTTCACAGAATCTATGGGAAATTCCTAGGCTTGAGAGCTTACATCAGAAAA

[0735] CAGATAAATAATATATTTTATAGGTTTATTTATGAAACAGAGCATCATAATGGCATAGCAGAGTTACTGG

[0736] AAATATTGGGAAGTATAATTAATGGATTTGCCTTACCACTAAAAGAAGAGCACAAGATTTTCTTATTGAA

[0737] GGTGTTACTACCTTTGCACAAAGTGAAATCTCTGAGTGTCTACCATCCCCAGCTGGCATACTGTGTAGTGC

[0738] AGTTTTTAGAAAAGGACAGCACCCTCACGGAACCAGTGGTGATGGCACTTCTCAAATACTGGCCAAAGAC

[0739] TCACAGTCCAAAAGAAGTAATGTTCTTAAACGAATTAGAAGAGATTTTAGATGTCATTGAACCATCAGAA

[0740] TTTGTGAAGATCATGGAACCCCTCTTCCGGCAGTTGGCCAAATGTGTCTCCAGCCCACACTTCCAGGTGGC

[0741] AGAGCGAGCTCTCTATTACTGGAATAATGAATACATCATGAGTTTAATCAGTGACAACGCAGCGAAGATT

[0742] CTGCCCATCATGTTTCCTTCCTTGTACCGCAACTCAAAGACCCATTGGAACAAGACAATACATGGCTTGAT

[0743] ATACAACGCCCTGAAGCTCTTCATGGAGATGAACCAAAAGCTATTTGATGACTGTACACAACAGTTCAAA

[0744] GCAGAGAAACTAAAAGAGAAGCTAAAAATGAAAGAACGGGAAGAAGCATGGGTTAAAATAGAAAATCT

[0745] AGCCAAAGCCAATCCCCAGTACACAGTGTATAGTCAAGCCAGCACCATGAGCATTCCGGTTGCAATGGAG

[0746] ACAGATGGGCCTTTATTTGAAGATGTGCAGATGCTGAGAAAGACAGTGAAGGACGAGGCTCATCAGGCA

[0747] CAGAAAGATCCGAAGAAGGACCGTCCTCTTGCACGCCGCAAGTCCGAGCTGCCTCAGGACCCCCACACC

[0748] AAGAAAGCCTTGGAAGCTCACTGCAGGGCCGATGAGCTGGCCTCCCAGGACGGCCGCTAG

[0749] SEQ ID NO: 39 > DNA sequence for human PP2A Catalytic subunit C alpha (PPP2CA):

[0750] ATGGACGAGAAGGTGTTCACCAAGGAGCTGGACCAGTGGATCGAGCAGCTGAACGAGTGCAAGCAGCTG

[0751] TCCGAGTCCCAGGTCAAGAGCCTCTGCGAGAAGGCTAAAGAAATCCTGACAAAAGAATCCAACGTGCAA

[0752] GAGGTTCGATGTCCAGTTACTGTCTGTGGAGATGTGCATGGGCAATTTCATGATCTCATGGAACTGTTTAG

[0753] AATTGGTGGCAAATCACCAGATACAAATTACTTGTTTATGGGAGATTATGTTGACAGAGGATATTATTCA

[0754] GTTGAAACAGTTACACTGCTTGTAGCTCTTAAGGTTCGTTACCGTGAACGCATCACCATTCTTCGAGGGAA

[0755] TCATGAGAGCAGACAGATCACACAAGTTTATGGTTTCTATGATGAATGTTTAAGAAAATATGGAAATGCA

[0756] AATGTTTGGAAATATTTTACAGATCTTTTTGACTATCTTCCTCTCACTGCCTTGGTGGATGGGCAGATCTTC

[0757] TGTCTACATGGTGGTCTCTCGCCATCTATAGATACACTGGATCATATCAGAGCACTTGATCGCCTACAAGA

[0758] AGTTCCCCATGAGGGTCCAATGTGTGACTTGCTGTGGTCAGATCCAGATGACCGTGGTGGTTGGGGTATA

[0759] TCTCCTCGAGGAGCTGGTTACACCTTTGGGCAAGATATTTCTGAGACATTTAATCATGCCAATGGCCTCAC

[0760] GTTGGTGTCTAGAGCTCACCAGCTAGTGATGGAGGGATATAACTGGTGCCATGACCGGAATGTAGTAACG

[0761] ATTTTCAGTGCTCCAAACTATTGTTATCGTTGTGGTAACCAAGCTGCAATCATGGAACTTGACGATACTCT

[0762] AAAATACTCTTTCTTGCAGTTTGACCCAGCACCTCGTAGAGGCGAGCCACATGTTACTCGTCGTACCCCAG

[0763] ACTACTTCCTGTAA

[0764] SEQ ID NO: 40 > DNA sequence for human Rec8 separase-cleavage target phospho-mimic (S / T to D substitutions): agaGACccaGACctcGATggctggctaccccctgaactactgggtctctggGACcattgtgcccagccacccccaaaagccctcaggcgagagctgcctgagga ggcagccgctgaggaggaaaggagaaagattgaagttccaGATgagattgaggtcccgagggaggccctggagcccGATgttccccttatggtgGATttagagat cGATctagaggcagctgaagaggagaagGACcgcatcGACctcatcccaccagaagaacggtgggcctggcctgaggtggaggcgccagaagctcctgcattgc ccgtggtgcctgaactccctgaggtgcccatggagatgcctttggtgctgcccccagagctcgagctgctcGATctggaagcagtgcacagggcagtggca

[0765] SEQ ID NO: 41 mmKi67 Mouse [Ki67 Chromatin Binding Domain] Protein Sequence

[0766] HSEKLAHDTSILKSTQQQKPDSVKPLRTCRRVLRASKEDPKEVLVDTRDHATLQSKSNPLLSPKRKSARDGSIV

[0767] RTRALRSLAPKQEATDEKPVPEKKRAASSKRHVSPEPVKMKHLKIVSNKLESVEEQVSTVMKTEEMEAKREN

[0768] PVTPDQNSRYRKKTNVKQPRPKFDASAENVGIKKNEKTMKTASQETELQNPDDGAKKSTSRGQVSGKRTCLR

[0769] SRGTTEMPQPCEAEEKTSKPAAEILIKPQEEKGVSGESDVRCLRSRKTRVALDSEPKPRVTRGTKKDAKTLKED

[0770] EDIVCTKKLRTRS

[0771] SEQ ID NO: 42 »Ki67 Mouse [Ki67 Chromatin Binding Domain] DNA Sequence

[0772] CACTCTGAGAAACTAGCACATGACACCAGTATCCTTAAGAGCACTCAACAGCAAAAGCCAGACTCAGTA AAACCTCTGAGAACATGCAGAAGAGTGCTGAGGGCCTCTAAAGAGGACCCCAAGGAAGTGTTGGTGGAC ACCAGAGACCATGCAACATTACAAAGCAAAAGCAACCCTTTGCTGTCCCCGAAGAGGAAGTCTGCAAGA GATGGAAGCATTGTGAGAACCAGGGCTTTGCGCTCTTTAGCACCAAAGCAGGAAGCAACAGATGAGAAG

[0773] CCTGTACCTGAGAAAAAAAGGGCTGCTTCCAGCAAGAGGCATGTATCACCTGAGCCTGTGAAGATGAAA

[0774] CACCTGAAAATCGTGTCAAACAAACTTGAATCTGTGGAAGAGCAGGTTAGCACTGTTATGAAAACAGAA GAAATGGAAGCCAAAAGAGAAAATCCTGTCACTCCAGATCAGAACTCTAGGTATCGAAAGAAAACCAAT GTAAAACAGCCAAGGCCCAAGTTTGATGCATCTGCAGAGAATGTCGGGATAAAGAAAAACGAGAAGACT ATGAAGACTGCCTCCCAGGAGACAGAGCTGCAGAATCCAGATGATGGAGCCAAGAAATCTACATCTCGG

[0775] GGCCAAGTCAGTGGGAAAAGAACATGCTTGAGGTCCAGAGGAACGACTGAGATGCCCCAGCCTTGTGAA GCAGAAGAGAAAACAAGCAAACCAGCTGCAGAAATCTTGATAAAGCCTCAGGAAGAGAAAGGAGTCTCT GGAGAGTCTGATGTTAGGTGTTTGAGGTCCAGAAAAACTAGAGTCGCTTTGGACAGTGAACCTAAGCCAA GGGTAACTCGTGGAACCAAGAAAGATGCAAAAACTCTGAAGGAGGATGAAGACATTGTATGCACCAAGA

[0776] AGTTAAGAACAAGAAGT

[0777] SEQ ID NO: 43 - rnEGFP Monomeric Enhanced Green Fluorescent Protein

[0778] Protein Sequence

[0779] VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSR

[0780] YPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY NSHNVYIMADKQKNGIKVNFKIRHNIED GS VQL ADHYQQNTPIGD GP VLLPDNHYL STQSKL SKDPNEKRDH MVLLEFVTAAGITLGMDELYK

[0781] SEQ ID NO: 44 rnEGFP Monomeric Enhanced Green Fluorescent Protein

[0782] DNA Sequence

[0783] GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAAC

[0784] GGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTC ATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGT GCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGT CCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG

[0785] CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA CAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAA GGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAA CACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGC

[0786] AAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG GCATGGACGAGCTGTACAAGTAA

[0787] SEQ ID NO: 45 mmKi67-mEGFP Mouse [Ki67 Chromatin Binding Domain] -[rnEGFP]

[0788] Protein Sequence

[0789] MDYKDHDGDYKDHDIDYKDDDDKHSEKLAHDTSILKSTQQQKPDSVKPLRTCRRVLRASKEDPKEVLVDTR

[0790] DHATLQSKSNPLLSPKRKSARDGSIVRTRALRSLAPKQEATDEKPVPEKKRAASSKRHVSPEPVKMKHLKIVS

[0791] NKLESVEEQVSTVMKTEEMEAKRENPVTPDQNSRYRKKTNVKQPRPKFDASAENVGIKKNEKTMKTASQETE

[0792] LQNPDDGAKKSTSRGQVSGKRTCLRSRGTTEMPQPCEAEEKTSKPAAEILIKPQEEKGVSGESDVRCLRSRKTR VALDSEPKPRVTRGTKKDAKTLKEDEDIVCTKKLRTRSEFVSKGEELFTGVVPILVELDGDVNGHKFSVSGEG EGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN YKTRAE VKFEGDTL VNRIELKGIDFKED GNIL GHKLEYNYNSHNVYIM ADKQKNGIKVNFKIRHNIED GS VQL

[0793] ADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0794] SEQ ID NO: 46 mmKi67-mEGFP Mouse [Ki67 Chromatin Binding Domain] -[rnEGFP]

[0795] DNA Sequence ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAG CACTCTGAGAAACTAGCACATGACACCAGTATCCTTAAGAGCACTCAACAGCAAAAGCCAGACTCAGTA AAACCTCTGAGAACATGCAGAAGAGTGCTGAGGGCCTCTAAAGAGGACCCCAAGGAAGTGTTGGTGGAC ACCAGAGACCATGCAACATTACAAAGCAAAAGCAACCCTTTGCTGTCCCCGAAGAGGAAGTCTGCAAGA GATGGAAGCATTGTGAGAACCAGGGCTTTGCGCTCTTTAGCACCAAAGCAGGAAGCAACAGATGAGAAG CCTGTACCTGAGAAAAAAAGGGCTGCTTCCAGCAAGAGGCATGTATCACCTGAGCCTGTGAAGATGAAA CACCTGAAAATCGTGTCAAACAAACTTGAATCTGTGGAAGAGCAGGTTAGCACTGTTATGAAAACAGAA GAAATGGAAGCCAAAAGAGAAAATCCTGTCACTCCAGATCAGAACTCTAGGTATCGAAAGAAAACCAAT GTAAAACAGCCAAGGCCCAAGTTTGATGCATCTGCAGAGAATGTCGGGATAAAGAAAAACGAGAAGACT ATGAAGACTGCCTCCCAGGAGACAGAGCTGCAGAATCCAGATGATGGAGCCAAGAAATCTACATCTCGG GGCCAAGTCAGTGGGAAAAGAACATGCTTGAGGTCCAGAGGAACGACTGAGATGCCCCAGCCTTGTGAA GCAGAAGAGAAAACAAGCAAACCAGCTGCAGAAATCTTGATAAAGCCTCAGGAAGAGAAAGGAGTCTCT GGAGAGTCTGATGTTAGGTGTTTGAGGTCCAGAAAAACTAGAGTCGCTTTGGACAGTGAACCTAAGCCAA GGGTAACTCGTGGAACCAAGAAAGATGCAAAAACTCTGAAGGAGGATGAAGACATTGTATGCACCAAGA

[0796] AGTTAAGAACAAGAAGTGAATTCGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGG TCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCT ACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGAC CACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAG TCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCC GCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGG AGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGC TCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCT GAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGT GACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0797] SEQ ID NO: 47 mmSecurin Mouse [SecurinfLPE)] Separase Cleavage Substrate (SCS) Protein Sequence

[0798] SSVPAPDDAYPEIEKFFPFNPLDFESFDLPEEHQISLLPLNGVPL

[0799] SEQ ID NO: 48 mmSecurin Mouse [Securin] Separase Cleavage Substrate (SCS)

[0800] DNA Sequence

[0801] AGTTCTGTTCCTGCTCCTGATGATGCCTACCCAGAAATAGAAAAGTTCTTCCCTTTCAATCCTCTCGACTTT GAGAGTTTTGACCTGCCTGAGGAGCACCAGATCTCACTTCTCCCCTTGAATGGCGTGCCTCTC

[0802] SEQ ID NO: 49 mmKi67-mmSecurin-mEGFP Mouse [Ki67 Chromatin Binding Domain] -[Securin-SRM]-[mEGFP] Protein Sequence

[0803] MHSEKLAHDTSILKSTQQQKPDSVKPLRTCRRVLRASKEDPKEVLVDTRDHATLQSKSNPLLSPKRKSARDGSI VRTRALRSLAPKQEATDEKPVPEKKRAASSKRHVSPEPVKMKHLKIVSNKLESVEEQVSTVMKTEEMEAKRE NPVTPDQNSRYRKKTNVKQPRPKFDASAENVGIKKNEKTMKTASQETELQNPDDGAKKSTSRGQVSGKRTCL RSRGTTEMPQPCEAEEKTSKPAAEILKPQEEKGVSGESDVRCLRSRKTRVALDSEPKPRVTRGTKKDAKTLKE DEDIVCTKKLRTRSEFSSVPAPDDAYPEIEKFFPFNPLDFESFDLPEEHQISLLPLNGVPLSRVSKGEELFTGVVPI LVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKS AMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQK NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITL GMDELYK

[0804] SEQ ID NO: 50 mmKi67-mmSecurin-mEGFP Mouse [Ki67 Chromatin Binding Domain]-[Securin-SRM]-[mEGFP] DNA Sequence

[0805] ATGCACTCTGAGAAACTAGCACATGACACCAGTATCCTTAAGAGCACTCAACAGCAAAAGCCAGACTCA GTAAAACCTCTGAGAACATGCAGAAGAGTGCTGAGGGCCTCTAAAGAGGACCCCAAGGAAGTGTTGGTG GACACCAGAGACCATGCAACATTACAAAGCAAAAGCAACCCTTTGCTGTCCCCGAAGAGGAAGTCTGCA AGAGATGGAAGCATTGTGAGAACCAGGGCTTTGCGCTCTTTAGCACCAAAGCAGGAAGCAACAGATGAG AAGCCTGTACCTGAGAAAAAAAGGGCTGCTTCCAGCAAGAGGCATGTATCACCTGAGCCTGTGAAGATG AAACACCTGAAAATCGTGTCAAACAAACTTGAATCTGTGGAAGAGCAGGTTAGCACTGTTATGAAAACA GAAGAAATGGAAGCCAAAAGAGAAAATCCTGTCACTCCAGATCAGAACTCTAGGTATCGAAAGAAAACC AATGTAAAACAGCCAAGGCCCAAGTTTGATGCATCTGCAGAGAATGTCGGGATAAAGAAAAACGAGAAG ACTATGAAGACTGCCTCCCAGGAGACAGAGCTGCAGAATCCAGATGATGGAGCCAAGAAATCTACATCT CGGGGCCAAGTCAGTGGGAAAAGAACATGCTTGAGGTCCAGAGGAACGACTGAGATGCCCCAGCCTTGT GAAGCAGAAGAGAAAACAAGCAAACCAGCTGCAGAAATCTTGATAAAGCCTCAGGAAGAGAAAGGAGT CTCTGGAGAGTCTGATGTTAGGTGTTTGAGGTCCAGAAAAACTAGAGTCGCTTTGGACAGTGAACCTAAG CCAAGGGTAACTCGTGGAACCAAGAAAGATGCAAAAACTCTGAAGGAGGATGAAGACATTGTATGCACC AAGAAGTTAAGAACAAGAAGTGAATTCAGTTCTGTTCCTGCTCCTGATGATGCCTACCCAGAAATAGAAA

[0806] AGTTCTTCCCTTTCAATCCTCTCGACTTTGAGAGTTTTGACCTGCCTGAGGAGCACCAGATCTCACTTCTCC CCTTGAATGGCGTGCCTCTCTCTAGAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCT GGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCAC CTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTG ACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCA AGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGAC CCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAA GGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGC

[0807] CGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCA GCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTAC CTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTC GTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG

[0808] SEQ ID NO: 51 mmRec8 Mouse [Rec8(C2;C3;LPE)] Separase Cleavage Substrate (SCS)

[0809] Protein Sequence

[0810] RTPTLSGWLPPELLGLWTHCAQVPQRMLRQRPQLETEETVEEERAADEEERRKTEALSEIEVLREAQEPSGPL

[0811] MLS SEL SLE AAEDEKSRTSLIPPEWWAWSEEGQPEPPALPMLPELPE VPLEMPPRPEL S SE AVLRAV A

[0812] SEQ ID NO: 52 mmRec8 Mouse [Rec8(C2;C3;LPE)] Separase Cleavage Substrate (SCS)

[0813] DNA Sequence

[0814] AGAACCCCAACTCTCTCTGGCTGGCTACCCCCAGAACTCCTAGGTTTATGGACACACTGTGCCCAGGTCCC

[0815] CCAAAGAATGCTCAGACAAAGGCCACAGCTGGAGACTGAGGAGACAGTGGAGGAAGAGAGAGCAGCAG

[0816] ATGAGGAAGAGAGGAGAAAGACTGAAGCTCTGAGCGAGATCGAGGTTCTGAGGGAGGCCCAGGAGCCC

[0817] AGTGGTCCCCTCATGCTGTCTTCAGAACTCTCCCTAGAAGCAGCAGAAGATGAAAAGTCTCGTACTAGCC TCATCCCCCCAGAATGGTGGGCCTGGTCTGAGGAGGGGCAGCCCGAGCCCCCTGCACTGCCCATGCTGCC TGAACTTCCTGAAGTGCCCTTGGAGATGCCCCCAAGGCCTGAACTCTCTTCAGAGGCTGTGCTCAGGGCA GTAGCA

[0818] SEQ ID NO: 53 mmKi67-mrnRec8-mEGFP Mouse [Ki67 Chromatin Binding Domain]-[Rec8-SCS]-[mEGFP] Protein Sequence

[0819] MHSEKLAHDTSILKSTQQQKPDSVKPLRTCRRVLRASKEDPKEVLVDTRDHATLQSKSNPLLSPKRKSARDGSI

[0820] VRTRALRSLAPKQEATDEKPVPEKKRAASSKRHVSPEPVKMKHLKIVSNKLESVEEQVSTVMKTEEMEAKRE

[0821] NPVTPDQNSRYRKKTNVKQPRPKFDASAENVGIKKNEKTMKTASQETELQNPDDGAKKSTSRGQVSGKRTCL

[0822] RSRGTTEMPQPCEAEEKTSKPAAEILKPQEEKGVSGESDVRCLRSRKTRVALDSEPKPRVTRGTKKDAKTLKE

[0823] DEDIVCTKKLRTRSEFRTPTLSGWLPPELLGLWTHCAQVPQRMLRQRPQLETEETVEEERAADEEERRKTEAL

[0824] SEIEVLREAQEPSGPLMLSSELSLEAAEDEKSRTSLIPPEWWAWSEEGQPEPPALPMLPELPEVPLEMPPRPELSS

[0825] EAVLRAVASRVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTT

[0826] LTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI LGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIED GS VQL ADHYQQNTPIGD GP VLLPDNHYL STQSKL S KDPNEKRDHMVLLEF VTA AGITL GMDEL YK

[0827] SEQ ID NO: 54 mmKi67-mmRec8-mEGFP Mouse [Ki67 Chromatin Binding Domain]-[Rec8-SCS]-[mEGFP] DNA Sequence

[0828] ATGCACTCTGAGAAACTAGCACATGACACCAGTATCCTTAAGAGCACTCAACAGCAAAAGCCAGACTCA

[0829] GTAAAACCTCTGAGAACATGCAGAAGAGTGCTGAGGGCCTCTAAAGAGGACCCCAAGGAAGTGTTGGTG

[0830] GACACCAGAGACCATGCAACATTACAAAGCAAAAGCAACCCTTTGCTGTCCCCGAAGAGGAAGTCTGCA

[0831] AGAGATGGAAGCATTGTGAGAACCAGGGCTTTGCGCTCTTTAGCACCAAAGCAGGAAGCAACAGATGAG

[0832] AAGCCTGTACCTGAGAAAAAAAGGGCTGCTTCCAGCAAGAGGCATGTATCACCTGAGCCTGTGAAGATG

[0833] AAACACCTGAAAATCGTGTCAAACAAACTTGAATCTGTGGAAGAGCAGGTTAGCACTGTTATGAAAACA GAAGAAATGGAAGCCAAAAGAGAAAATCCTGTCACTCCAGATCAGAACTCTAGGTATCGAAAGAAAACC AATGTAAAACAGCCAAGGCCCAAGTTTGATGCATCTGCAGAGAATGTCGGGATAAAGAAAAACGAGAAG ACTATGAAGACTGCCTCCCAGGAGACAGAGCTGCAGAATCCAGATGATGGAGCCAAGAAATCTACATCT

[0834] CGGGGCCAAGTCAGTGGGAAAAGAACATGCTTGAGGTCCAGAGGAACGACTGAGATGCCCCAGCCTTGT

[0835] GAAGCAGAAGAGAAAACAAGCAAACCAGCTGCAGAAATCTTGATAAAGCCTCAGGAAGAGAAAGGAGT CTCTGGAGAGTCTGATGTTAGGTGTTTGAGGTCCAGAAAAACTAGAGTCGCTTTGGACAGTGAACCTAAG CCAAGGGTAACTCGTGGAACCAAGAAAGATGCAAAAACTCTGAAGGAGGATGAAGACATTGTATGCACC AAGAAGTTAAGAACAAGAAGTGAATTCAGAACCCCAACTCTCTCTGGCTGGCTACCCCCAGAACTCCTAG

[0836] GTTTATGGACACACTGTGCCCAGGTCCCCCAAAGAATGCTCAGACAAAGGCCACAGCTGGAGACTGAGG AGACAGTGGAGGAAGAGAGAGCAGCAGATGAGGAAGAGAGGAGAAAGACTGAAGCTCTGAGCGAGATC GAGGTTCTGAGGGAGGCCCAGGAGCCCAGTGGTCCCCTCATGCTGTCTTCAGAACTCTCCCTAGAAGCAG CAGAAGATGAAAAGTCTCGTACTAGCCTCATCCCCCCAGAATGGTGGGCCTGGTCTGAGGAGGGGCAGC

[0837] CCGAGCCCCCTGCACTGCCCATGCTGCCTGAACTTCCTGAAGTGCCCTTGGAGATGCCCCCAAGGCCTGA

[0838] ACTCTCTTCAGAGGCTGTGCTCAGGGCAGTAGCATCTAGAGTGAGCAAGGGCGAGGAGCTGTTCACCGGG

[0839] GTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGC

[0840] GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCT

[0841] GGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCA

[0842] GCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGAC

[0843] GGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG

[0844] GGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAG GACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGC CCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGG TCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG

[0845] SEQ ID NO: 55 mmMeikin Mouse [Meikin] Separase Cleavage Substrate (SCS)

[0846] Protein Sequence

[0847] SCLPSCSNAELHIESTDERGSSFPSPELFRGSDCLDWEHPKLEDYMFYKNSTLLDTSKAVV

[0848] SEQ ID NO: 56 mmMeikin Mouse [Meikin] Separase Cleavage Substrate (SCS)DNA Sequence

[0849] AGCTGTCTCCCGAGTTGTTCAAATGCAGAGTTACATACAGAGAGCACCGATGAGAGAGGAAGTAGCTTCC CATCGCCAGAACTGTTCAGAGGGTCAGACTGCCTGGATTGGGAGCATCCCAAGTTGGAAGATTACATGTT CTACAAGAACTCTACTCTTTTAGACACCAGTAAAGCAGTAGTT

[0850] SEQ ID NO: 57 mmKi67-mmMeikin-mEGFP Mouse [Ki67 Chromatin Binding Domain]-[Meikin-SCS]-[mEGFP] Protein Sequence

[0851] MHSEKLAHDTSILKSTQQQKPDSVKPLRTCRRVLRASKEDPKEVLVDTRDHATLQSKSNPLLSPKRKSARDGSI

[0852] VRTRALRSLAPKQEATDEKPVPEKKRAASSKRHVSPEPVKMKHLKIVSNKLESVEEQVSTVMKTEEMEAKRE

[0853] NPVTPDQNSRYRKKTNVKQPRPKFDASAENVGIKKNEKTMKTASQETELQNPDDGAKKSTSRGQVSGKRTCL

[0854] RSRGTTEMPQPCEAEEKTSKPAAEILKPQEEKGVSGESDVRCLRSRKTRVALDSEPKPRVTRGTKKDAKTLKE DEDIVCTKKLRTRSEFSCLPSCSNAELHTESTDERGSSFPSPELFRGSDCLDWEHPKLEDYMFYKNSTLLDTSKA VVSRVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQ CFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLE

[0855] YNYNSHNVYIMADKQKNGIKVNFKIRHNIED GS VQL ADH YQQNTPIGD GP VLLPDNHYL STQSKL SKDPNEK RDHM VLLEF VT A AGITL GMDEL YK

[0856] SEQ ID NO: 58 mmKi67-mmMeikin-mEGFP Mouse [Ki67 Chromatin Binding Domain]-[Meikin-SCS]-[mEGFP] DNA Sequence

[0857] ATGCACTCTGAGAAACTAGCACATGACACCAGTATCCTTAAGAGCACTCAACAGCAAAAGCCAGACTCA GTAAAACCTCTGAGAACATGCAGAAGAGTGCTGAGGGCCTCTAAAGAGGACCCCAAGGAAGTGTTGGTG GACACCAGAGACCATGCAACATTACAAAGCAAAAGCAACCCTTTGCTGTCCCCGAAGAGGAAGTCTGCA AGAGATGGAAGCATTGTGAGAACCAGGGCTTTGCGCTCTTTAGCACCAAAGCAGGAAGCAACAGATGAG

[0858] AAGCCTGTACCTGAGAAAAAAAGGGCTGCTTCCAGCAAGAGGCATGTATCACCTGAGCCTGTGAAGATG AAACACCTGAAAATCGTGTCAAACAAACTTGAATCTGTGGAAGAGCAGGTTAGCACTGTTATGAAAACA GAAGAAATGGAAGCCAAAAGAGAAAATCCTGTCACTCCAGATCAGAACTCTAGGTATCGAAAGAAAACC

[0859] AATGTAAAACAGCCAAGGCCCAAGTTTGATGCATCTGCAGAGAATGTCGGGATAAAGAAAAACGAGAAG ACTATGAAGACTGCCTCCCAGGAGACAGAGCTGCAGAATCCAGATGATGGAGCCAAGAAATCTACATCT CGGGGCCAAGTCAGTGGGAAAAGAACATGCTTGAGGTCCAGAGGAACGACTGAGATGCCCCAGCCTTGT GAAGCAGAAGAGAAAACAAGCAAACCAGCTGCAGAAATCTTGATAAAGCCTCAGGAAGAGAAAGGAGT

[0860] CTCTGGAGAGTCTGATGTTAGGTGTTTGAGGTCCAGAAAAACTAGAGTCGCTTTGGACAGTGAACCTAAG CCAAGGGTAACTCGTGGAACCAAGAAAGATGCAAAAACTCTGAAGGAGGATGAAGACATTGTATGCACC AAGAAGTTAAGAACAAGAAGTGAATTCAGCTGTCTCCCGAGTTGTTCAAATGCAGAGTTACATACAGAG AGCACCGATGAGAGAGGAAGTAGCTTCCCATCGCCAGAACTGTTCAGAGGGTCAGACTGCCTGGATTGG

[0861] GAGCATCCCAAGTTGGAAGATTACATGTTCTACAAGAACTCTACTCTTTTAGACACCAGTAAAGCAGTAG TTTCTAGAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGA CGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCT GAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGC

[0862] GTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAG GCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTT CGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCT GGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGG

[0863] CATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCA GCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAG CTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATC ACTCTCGGCATGGACGAGCTGTACAAG

[0864] SEQ ID NO: 59 mmRec8S / T>D Mouse [Rec8(C2;C3;LPE); S / T>D] Phosphomimetic Separase Cleavage Substrate (SCS) (S / T>D mutations underlined) Protein Sequence

[0865] RDPDLDGWLPPELLGLWDHCAQVPQRMLRQRPQLEDEEDVEEERAADEEERRKDEALDEIEVLREAQEPDGP LMLDDELDLEAAEDEKDRDDLIPPEWWAWDEEGQPEPPALPMLPELPEVPLEMPPRPELDDEAVLRAVA

[0866] SEQ ID NO: 60 mmRec8S / T>D Mouse [Rec8(C2;C3;LPE); S / T>D] Phosphomimetic Separase Cleavage Substrate (SCS) (S / T>D mutations underlined) DNA Sequence

[0867] AGAGATCCAGATCTCGATGGCTGGCTACCCCCAGAACTCCTAGGTTTATGGGACCACTGTGCCCAGGTCC CCCAAAGAATGCTCAGACAAAGGCCACAGCTGGAGGATGAGGAGGACGTGGAGGAAGAGAGAGCAGCA GATGAGGAAGAGAGGAGAAAGGACGAAGCTCTGGATGAGATCGAGGTTCTGAGGGAGGCCCAGGAGCC CGATGGTCCCCTCATGCTGGATGACGAACTCGACCTAGAAGCAGCAGAAGATGAAAAGGATCGTGATGA

[0868] CCTCATCCCCCCAGAATGGTGGGCCTGGGATGAGGAGGGGCAGCCCGAGCCCCCTGCACTGCCCATGCTG CCTGAACTTCCTGAAGTGCCCTTGGAGATGCCCCCAAGGCCTGAACTCGATGACGAGGCTGTGCTCAGGG CAGTAGCA

[0869] SEQ ID NO: 61 mmKi67-mmRec8S / T>D-mEGFP Mouse [Ki67 Chromatin Binding Domain]-[mmRec8S / T>D-SCS]- [inEGFP] Protein Sequence

[0870] MHSEKLAHDTSILKSTQQQKPDSVKPLRTCRRVLRASKEDPKEVLVDTRDHATLQSKSNPLLSPKRKSARDGSI

[0871] VRTRALRSLAPKQEATDEKPVPEKKRAASSKRHVSPEPVKMKHLKIVSNKLESVEEQVSTVMKTEEMEAKRE

[0872] NPVTPDQNSRYRKKTNVKQPRPKFDASAENVGIKKNEKTMKTASQETELQNPDDGAKKSTSRGQVSGKRTCL

[0873] RSRGTTEMPQPCEAEEKTSKPAAEILKPQEEKGVSGESDVRCLRSRKTRVALDSEPKPRVTRGTKKDAKTLKE

[0874] DEDIVCTKKLRTRSEFRDPDLDGWLPPELLGLWDHCAQVPQRMLRQRPQLEDEEDVEEERAADEEERRKDEA

[0875] LDEIEVLREAQEPDGPLMLDDELDLEAAEDEKDRDDLIPPEWWAWDEEGQPEPPALPMLPELPEVPLEMPPRP

[0876] ELDDEAVLRAVASRVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT

[0877] LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

[0878] DGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQS KLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0879] SEQ ID NO: 62 - mmKi67-mmRec8S / T>D-mEGFP Mouse [Ki67 Chromatin Binding Domain]-[mmRec8S / T>D-SCS]-

[0880] [mEGFP] DNA Sequence

[0881] ATGCACTCTGAGAAACTAGCACATGACACCAGTATCCTTAAGAGCACTCAACAGCAAAAGCCAGACTCA

[0882] GTAAAACCTCTGAGAACATGCAGAAGAGTGCTGAGGGCCTCTAAAGAGGACCCCAAGGAAGTGTTGGTG

[0883] GACACCAGAGACCATGCAACATTACAAAGCAAAAGCAACCCTTTGCTGTCCCCGAAGAGGAAGTCTGCA

[0884] AGAGATGGAAGCATTGTGAGAACCAGGGCTTTGCGCTCTTTAGCACCAAAGCAGGAAGCAACAGATGAG

[0885] AAGCCTGTACCTGAGAAAAAAAGGGCTGCTTCCAGCAAGAGGCATGTATCACCTGAGCCTGTGAAGATG

[0886] AAACACCTGAAAATCGTGTCAAACAAACTTGAATCTGTGGAAGAGCAGGTTAGCACTGTTATGAAAACA

[0887] GAAGAAATGGAAGCCAAAAGAGAAAATCCTGTCACTCCAGATCAGAACTCTAGGTATCGAAAGAAAACC

[0888] AATGTAAAACAGCCAAGGCCCAAGTTTGATGCATCTGCAGAGAATGTCGGGATAAAGAAAAACGAGAAG

[0889] ACTATGAAGACTGCCTCCCAGGAGACAGAGCTGCAGAATCCAGATGATGGAGCCAAGAAATCTACATCT

[0890] CGGGGCCAAGTCAGTGGGAAAAGAACATGCTTGAGGTCCAGAGGAACGACTGAGATGCCCCAGCCTTGT

[0891] GAAGCAGAAGAGAAAACAAGCAAACCAGCTGCAGAAATCTTGATAAAGCCTCAGGAAGAGAAAGGAGT

[0892] CTCTGGAGAGTCTGATGTTAGGTGTTTGAGGTCCAGAAAAACTAGAGTCGCTTTGGACAGTGAACCTAAG

[0893] CCAAGGGTAACTCGTGGAACCAAGAAAGATGCAAAAACTCTGAAGGAGGATGAAGACATTGTATGCACC

[0894] AAGAAGTTAAGAACAAGAAGTGAATTCAGAGATCCAGATCTCGATGGCTGGCTACCCCCAGAACTCCTA

[0895] GGTTTATGGGACCACTGTGCCCAGGTCCCCCAAAGAATGCTCAGACAAAGGCCACAGCTGGAGGATGAG

[0896] GAGGACGTGGAGGAAGAGAGAGCAGCAGATGAGGAAGAGAGGAGAAAGGACGAAGCTCTGGATGAGAT

[0897] CGAGGTTCTGAGGGAGGCCCAGGAGCCCGATGGTCCCCTCATGCTGGATGACGAACTCGACCTAGAAGC

[0898] AGCAGAAGATGAAAAGGATCGTGATGACCTCATCCCCCCAGAATGGTGGGCCTGGGATGAGGAGGGGCA

[0899] GCCCGAGCCCCCTGCACTGCCCATGCTGCCTGAACTTCCTGAAGTGCCCTTGGAGATGCCCCCAAGGCCT

[0900] GAACTCGATGACGAGGCTGTGCTCAGGGCAGTAGCATCTAGAGTGAGCAAGGGCGAGGAGCTGTTCACC

[0901] GGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAG

[0902] GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGC

[0903] CCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAA

[0904] GCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGAC

[0905] GACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTG

[0906] AAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCAC

[0907] AACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATC

[0908] GAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTG

[0909] CTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCAC ATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG

[0910] SEQ ID NO: 63 - 3xFLAG-hKi67-hRec8-mEGFP Human [Ki67 Chromatin Binding Domain] - [Rec8(C2;C3;LPE)] - [mEGFP] Protein Sequence used for mRNA expression

[0911] MADYKDHDGDYKDHDIDYKDDDDKGTHTEELANGAADSFTSAPKQTPDSGKPLKISRRVLRAPKVEPVGDV

[0912] VSTRDPVKSQSKSNTSLPPLPFKRGGGKDGSVTGTKRLRCMPAPEEIVEELPASKKQRVAPRARGKSSEPVVIM

[0913] KRSLRTSAKRIEPAEELNSNDMKTNKEEHKLQDSVPENKGISLRSRRQNKTEAEQQITEVFVLAERIEINRNEKK

[0914] PMKTSPEMDIQNPDDGARKPIPRDKVTENKRCLRSARQNESSQPKVAEESGGQKSAKVLMQNQKGKGEAGNS

[0915] DSMCLRSRKTKSQPAASTLESKSVQRVTRSVKRCAENPKKAEDNVCVKKIRTRSHRDSEDIGSRTPTLSGWLP

[0916] PELLGLWTHCAQPPPKALRRELPEEAAAEEERRKIEVPSEIEVPREALEPSVPLMVSLEISLEAAEEEKSRISLIPP EERWAWPEVEAPEAPALPVVPELPEVPMEMPLVLPPELELLSLEAVHRAVAEFHDSRVSKGEELFTGVVPILVE

[0917] LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMP

[0918] EGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIED GS VQL ADHYQQNTPIGD GP VLLPDNHYL STQSKL SKDPNEKRDHMVLLEFVTA AGITL GMDE LYK SEQ ID NO: 64 - 3xFLAG-hKi67-hRec8-mEGFP Human [Ki67 Chromatin Binding Domain] - [Rec8(C2;C3;LPE)] - [rnEGFP] DNA Sequence used for mRNA expression

[0919] ATGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC

[0920] AAGGGTACCCACACTGAGGAACTGGCAAATGGTGCTGCTGATAGCTTTACAAGCGCTCCAAAGCAAACA

[0921] CCTGACAGTGGAAAACCTCTAAAAATATCCAGAAGAGTTCTTCGGGCCCCTAAAGTAGAACCCGTGGGA GACGTGGTAAGCACCAGAGACCCTGTAAAATCACAAAGCAAAAGCAACACTTCCCTGCCCCCACTGCCCT

[0922] TCAAGAGGGGAGGTGGCAAAGATGGAAGCGTCACGGGAACCAAGAGGCTGCGCTGCATGCCAGCACCA

[0923] GAGGAAATTGTGGAGGAGCTGCCAGCCAGCAAGAAGCAGAGGGTTGCTCCCAGGGCAAGAGGCAAATC

[0924] ATCCGAACCCGTGGTCATCATGAAGAGAAGTTTGAGGACTTCTGCAAAAAGAATTGAACCTGCGGAAGA GCTGAACAGCAACGACATGAAAACCAACAAAGAGGAACACAAATTACAAGACTCGGTCCCTGAAAATAA

[0925] GGGAATATCCCTGCGCTCCAGACGCCAAAATAAGACTGAGGCAGAACAGCAAATAACTGAGGTCTTTGT

[0926] ATTAGCAGAAAGAATAGAAATAAACAGAAATGAAAAGAAGCCCATGAAGACCTCCCCAGAGATGGACA

[0927] TTCAGAATCCAGATGATGGAGCCCGGAAACCCATACCTAGAGACAAAGTCACTGAGAACAAAAGGTGCT

[0928] TGAGGTCTGCTAGACAGAATGAGAGCTCCCAGCCTAAGGTGGCAGAGGAGAGCGGAGGGCAGAAGAGT

[0929] GCGAAGGTTCTCATGCAGAATCAGAAAGGGAAAGGAGAAGCAGGAAATTCAGACTCCATGTGCCTGAGA

[0930] TCAAGAAAGACAAAAAGCCAGCCTGCAGCAAGCACTTTGGAGAGCAAATCTGTGCAGAGAGTAACGCGG

[0931] AGTGTCAAGAGGTGTGCAGAAAATCCAAAGAAGGCTGAGGACAATGTGTGTGTCAAGAAAATAAGAACC

[0932] AGAAGTCATAGGGACAGTGAAGATATTGGATCCAGAACCCCAACTCTCTCTGGCTGGCTACCCCCTGAAC

[0933] TACTGGGTCTCTGGACCCATTGTGCCCAGCCACCCCCAAAAGCCCTCAGGCGAGAGCTGCCTGAGGAGGC AGCCGCTGAGGAGGAAAGGAGAAAGATTGAAGTTCCAAGTGAGATTGAGGTCCCGAGGGAGGCCCTGGA GCCCAGTGTTCCCCTTATGGTGTCTTTAGAGATCTCCCTAGAGGCAGCTGAAGAGGAGAAGTCCCGCATC

[0934] AGCCTCATCCCACCAGAAGAACGGTGGGCCTGGCCTGAGGTGGAGGCGCCAGAAGCTCCTGCATTGCCC GTGGTGCCTGAACTCCCTGAGGTGCCCATGGAGATGCCTTTGGTGCTGCCCCCAGAGCTCGAGCTGCTCTC

[0935] ACTGGAAGCAGTGCACAGGGCAGTGGCAGAATTCCACGATTCTAGAGTGAGCAAGGGCGAGGAGCTGTT CACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGC GAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCC GTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACA

[0936] TGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAA GGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGA

[0937] GCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAG

[0938] CCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAA

[0939] CATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGT GCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGA TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG

[0940] SEQ ID NO: 65 - 3xHis-hKi67-hRec8-mEGFP Human [Ki67 Chromatin Binding Domain] - [Rec8(C2;C3;LPE)] - [rnEGFP] Protein Sequence used for protein expression

[0941] MHTEELANGAADSFTSAPKQTPDSGKPLKISRRVLRAPKVEPVGDVVSTRDPVKSQSKSNTSLPPLPFKRGGG

[0942] KDGSVTGTKRLRCMPAPEEIVEELPASKKQRVAPRARGKSSEPVVIMKRSLRTSAKRIEPAEELNSNDMKTNK

[0943] EEHKLQDSVPENKGISLRSRRQNKTEAEQQITEVFVLAERIEINRNEKKPMKTSPEMDIQNPDDGARKPIPRDK

[0944] VTENKRCLRSARQNESSQPKVAEESGGQKSAKVLMQNQKGKGEAGNSDSMCLRSRKTKSQPAASTLESKSV

[0945] QRVTRSVKRCAENPKKAEDNVCVKKIRTRSHRDSEDIRTPTLSGWLPPELLGLWTHCAQPPPKALRRELPEEA AAEEERRKIEVPSEIEVPREALEPSVPLMVSLEISLEAAEEEKSRISLIPPEERWAWPEVEAPEAPALPVVPELPEV

[0946] PMEMPLVLPPELELLSLEAVHRAVAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFIC

[0947] TTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV NRIELKGIDFKED GNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIED GS VQL ADH YQQNTPIGD GP V LLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0948] SEQ ID NO: 66 - 3xHis-hKi67-hRec8-mEGFP Human [Ki67 Chromatin Binding Domain] - [Rec8(C2;C3;LPE)] - [rnEGFP] DNA Sequence used for protein expression

[0949] ATGCATCATCACCATCACCACCACACTGAGGAACTGGCAAATGGTGCTGCTGATAGCTTTACAAGCGCTC

[0950] CAAAGCAAACACCTGACAGTGGAAAACCTCTAAAAATATCCAGAAGAGTTCTTCGGGCCCCTAAAGTAG

[0951] AACCCGTGGGAGACGTGGTAAGCACCAGAGACCCTGTAAAATCACAAAGCAAAAGCAACACTTCCCTGC CCCCACTGCCCTTCAAGAGGGGAGGTGGCAAAGATGGAAGCGTCACGGGAACCAAGAGGCTGCGCTGCA

[0952] TGCCAGCACCAGAGGAAATTGTGGAGGAGCTGCCAGCCAGCAAGAAGCAGAGGGTTGCTCCCAGGGCAA

[0953] GAGGCAAATCATCCGAACCCGTGGTCATCATGAAGAGAAGTTTGAGGACTTCTGCAAAAAGAATTGAAC

[0954] CTGCGGAAGAGCTGAACAGCAACGACATGAAAACCAACAAAGAGGAACACAAATTACAAGACTCGGTCC

[0955] CTGAAAATAAGGGAATATCCCTGCGCTCCAGACGCCAAAATAAGACTGAGGCAGAACAGCAAATAACTG

[0956] AGGTCTTTGTATTAGCAGAAAGAATAGAAATAAACAGAAATGAAAAGAAGCCCATGAAGACCTCCCCAG

[0957] AGATGGACATTCAGAATCCAGATGATGGAGCCCGGAAACCCATACCTAGAGACAAAGTCACTGAGAACA AAAGGTGCTTGAGGTCTGCTAGACAGAATGAGAGCTCCCAGCCTAAGGTGGCAGAGGAGAGCGGAGGGC

[0958] AGAAGAGTGCGAAGGTTCTCATGCAGAATCAGAAAGGGAAAGGAGAAGCAGGAAATTCAGACTCCATGT GCCTGAGATCAAGAAAGACAAAAAGCCAGCCTGCAGCAAGCACTTTGGAGAGCAAATCTGTGCAGAGAG TAACGCGGAGTGTCAAGAGGTGTGCAGAAAATCCAAAGAAGGCTGAGGACAATGTGTGTGTCAAGAAAA TAAGAACCAGAAGTCATAGGGACAGTGAAGATATTAGAACCCCAACTCTCTCTGGCTGGCTACCCCCTGA

[0959] ACTACTGGGTCTCTGGACCCATTGTGCCCAGCCACCCCCAAAAGCCCTCAGGCGAGAGCTGCCTGAGGAG GCAGCCGCTGAGGAGGAAAGGAGAAAGATTGAAGTTCCAAGTGAGATTGAGGTCCCGAGGGAGGCCCTG GAGCCCAGTGTTCCCCTTATGGTGTCTTTAGAGATCTCCCTAGAGGCAGCTGAAGAGGAGAAGTCCCGCA TCAGCCTCATCCCACCAGAAGAACGGTGGGCCTGGCCTGAGGTGGAGGCGCCAGAAGCTCCTGCATTGCC CGTGGTGCCTGAACTCCCTGAGGTGCCCATGGAGATGCCTTTGGTGCTGCCCCCAGAGCTCGAGCTGCTCT CACTGGAAGCAGTGCACAGGGCAGTGGCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCA TCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT CGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTC TTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACA AGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACT TCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCA TGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCG TGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA CTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGA GTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0960] SEQ ID NO: 68 - 3xFLAG Tag Protein Sequence

[0961] MADYKDHDGDYKDHDIDYKDDDDK

[0962] SEQ ID NO: 69 - 3xFLAG Tag DNA Sequence

[0963] ATGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC AAG

[0964] SEQ ID NO: 70 - 6xHIS Tag Protein Sequence

[0965] MHHHHHH

[0966] SEQ ID NO: 71 - 6xHIS Tag DNA Sequence

[0967] ATGCATCATCACCATCACCAC

[0968] SEQ ID NO: 72 - UPTAL[mmCentro] Mouse Centromere Transcription Activator-Like (TAL) Protein Sequence VDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVTYQHIITALPEATHEDI VGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGVTAMEAVHASRNALTGAPLNLTPDQVVA IASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGK QALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALE TVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPV LCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGRPALESIVAQLSRPDPALAALT NDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRVA

[0969] SEQ ID NO: 73 - UPTAL[mmCentro] Mouse Centromere Transcription Activator-Like (TAL) DNA Sequence GTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACA GTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAAC ACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGAGGCGACACA CGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTCACGGATGCG GGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAACGTGGCGGC GTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAACCTGACCCCTG ATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCC AGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAACAATGGTGGCAAA CAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCAAG TTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTCTACCAGTTCTC TGTCAAGCCCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTC TTGAGACCGTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGC GATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAG CCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAAC CGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCT AGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTGTCAAGCCCACG GACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCA ACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAAT GGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTTCCAGTTCTTTGTCAAGCTCACGGACTCA CCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCT TCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTCATGACGGT GGCAAACAGGCCCTTGAGACAGTCCAGCGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTG ATCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCC AGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAA CAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGT TGTAGCGATTGCTAGTAACAATGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTC TGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGCGGTCGACCGGCGC TGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAACGACCACCTCGTC GCCTTGGCCTGCCTCGGCGGACGTCCTGCGATGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGAAT TGATCCGTAGAGTCAATCGCCGTATTGGCGAACGCACATCCCATCGCGTTGCC

[0970] SEQ ID NO: 75 - 3xmmSgo2CC 3x Mouse Shugoshin-2 Coiled-Coil Protein Sequence (with linker)

[0971] GGGGSGGGGSIFKISLKHNNRALARALSKEKENSRRITTEKMQLQKEVEKLNFENTGGGGSIFKISLKHNNRAL ARAL SKEKENSRRITIEKMQLQKEVEKLNFENTGGGGSIFKISLKHNNRAL ARAL SKEKENSRRITTEKMQLQ KEVEKLNFENT

[0972] SEQ ID NO: 75 - 3xmmSgo2CC 3x Mouse Shugoshin-2 Coiled-Coil Protein Sequence (without N-terminal linker) IFKISLKHNNRALARALSKEKENSRRITTEKMQLQKEVEKLNFENTGGGGSIFKISLKHNNRALARALSKEKEN SRRITTEKMQLQKEVEKLNFENTGGGGSIFKISLKHNNRALARALSKEKENSRRITTEKMQLQKEVEKLNFENT

[0973] SEQ ID NO: 76 - 3xmmSgo2CC 3x Mouse Shugoshin-2 Coiled-Coil DNA Sequence

[0974] GGTGGAGGCGGAAGTGGTGGAGGCGGAAGTATTTTTAAAATTAGCCTGAAACATAACAACCGCGCGCTG GCGCGCGCGCTGAGCAAAGAAAAAGAAAACAGCCGCCGCATTACCACCGAAAAAATGCAGCTGCAGAA AGAAGTGGAAAAACTGAACTTTGAAAACACTGGTGGAGGCGGAAGTATTTTTAAAATTAGCCTGAAACA TAACAACCGCGCGCTGGCGCGCGCGCTGAGCAAAGAAAAAGAAAACAGCCGCCGCATTACCACCGAAAA AATGCAGCTGCAGAAAGAAGTGGAAAAACTGAACTTTGAAAACACTGGTGGAGGCGGAAGTATTTTTAA AATTAGCCTGAAACATAACAACCGCGCGCTGGCGCGCGCGCTGAGCAAAGAAAAAGAAAACAGCCGCCG CATTACCACCGAAAAAATGCAGCTGCAGAAAGAAGTGGAAAAACTGAACTTTGAAAACACT

[0975] SEQ ID NO: 77 - 3xFLAG-UPTAL[mmCentro]-mEGFP-3xmmSgo2CC Mouse Centromere TAL-mEGFP-3x Mouse Shugoshin-2 Coiled-Coil Protein Sequence

[0976] MADYKDHDGDYKDHDIDYKDDDDKGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL SQHPAALGTVAVTYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGV

[0977] TAMEAVHASRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQA LETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETV

[0978] QRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLP VLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQ

[0979] AHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGL TPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAI

[0980] ASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRV AGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC FSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY

[0981] NYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKR DHM VLLEFVTA AGITL GMDEL YKEFGGGGS GGGGSIFKISLKHNNRAL ARAL SKEKENSRRITTEKMQLQKEV

[0982] EKLNFENTGGGGSIFKISLKHNNRALARALSKEKENSRRITTEKMQLQKEVEKLNFENTGGGGSIFKISLKHNN RAL ARAL SKEKENSRRITIEKMQLQKE VEKLNFENT

[0983] SEQ ID NO: 78 - 3xFLAG-UPTAL[mmCentro]-mEGFP-3xmmSgo2CC Mouse Centromere TAL-mEGFP-3x Mouse Shugoshin-2 Coiled-Coil DNA Sequence

[0984] ATGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC AAGGGTACCGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTT CGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGC TCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGA GGCGACACACGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTC ACGGATGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAA CGTGGCGGCGTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAAC CTGACCCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAAC

[0985] GACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAACAA TGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACT CCTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTC TACCAGTTCTCTGTCAAGCCCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGG CAAACAGGCTCTTGAGACCGTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCG CAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAG TTCTTTGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACA GGCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTT

[0986] GTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTG TCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTT GAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGA TTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTTCCAGTTCTTTGTCAAGC TCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACC GTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTA GTCATGACGGTGGCAAACAGGCCCTTGAGACAGTCCAGCGACTCCTCCCAGTTCTCTGTCAAGCCCACGG ACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAG CGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGA CGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACC CCAGATCAAGTTGTAGCGATTGCTAGTAACAATGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCC

[0987] TCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGCGG TCGACCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAAC GACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGATGGATGCAGTGAAAAAGGGATTGCCGC

[0988] ACGCGCCGGAATTGATCCGTAGAGTCAATCGCCGTATTGGCGAACGCACATCCCATCGCGTTGCCGGATC CGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAA CGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAG

[0989] TGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGC

[0990] ACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCA AGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGA

[0991] ACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAG CAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTC GGCATGGACGAGCTGTACAAGGAATTCGGTGGAGGCGGAAGTGGTGGAGGCGGAAGTATTTTTAAAATT

[0992] AGCCTGAAACATAACAACCGCGCGCTGGCGCGCGCGCTGAGCAAAGAAAAAGAAAACAGCCGCCGCATT

[0993] ACCACCGAAAAAATGCAGCTGCAGAAAGAAGTGGAAAAACTGAACTTTGAAAACACTGGTGGAGGCGG AAGTATTTTTAAAATTAGCCTGAAACATAACAACCGCGCGCTGGCGCGCGCGCTGAGCAAAGAAAAAGA

[0994] AAACAGCCGCCGCATTACCACCGAAAAAATGCAGCTGCAGAAAGAAGTGGAAAAACTGAACTTTGAAAA CACTGGTGGAGGCGGAAGTATTTTTAAAATTAGCCTGAAACATAACAACCGCGCGCTGGCGCGCGCGCTG

[0995] AGCAAAGAAAAAGAAAACAGCCGCCGCATTACCACCGAAAAAATGCAGCTGCAGAAAGAAGTGGAAAA ACTGAACTTTGAAAACACT

[0996] SEQ ID NO: 79 - 3xmmSTRN3CC 3x Mouse Striatin-3 Coiled-Coil Protein Sequence

[0997] GGGGSGGGGSWEVERAELQARIAFLQGERKGQENLKKDLVRRIKMLEYGGGGSWEVERAELQARIAFLQGE

[0998] RKGQENLKKDLVRRIKMLEYGGGGSWEVERAELQARIAFLQGERKGQENLKKDLVRRIKMLEY

[0999] SEQ ID NO: 80 - 3xmmSTRN3CC 3x Mouse Striatin-3 Coiled-Coil DNA Sequence

[1000] GGTGGAGGCGGAAGTGGTGGAGGCGGAAGTTGGGAAGTGGAACGCGCGGAACTGCAGGCGCGCATTGC GTTTCTGCAGGGCGAACGCAAAGGCCAGGAAAACCTGAAAAAAGATCTGGTGCGCCGCATTAAAATGCT

[1001] GGAATATGGTGGTGGGGGTAGCTGGGAGGTCGAGAGAGCCGAATTGCAAGCCCGAATTGCCTTCCTTCA GGGAGAACGCAAGGGGCAAGAGAACTTGAAAAAAGACTTGGTCCGCCGCATAAAAATGCTTGAATATGG

[1002] TGGTGGAGGTTCATGGGAGGTTGAACGTGCCGAACTGCAAGCTCGCATTGCGTTCTTACAAGGAGAACGC AAGGGTCAGGAAAACTTAAAAAAGGACCTTGTGCGTCGCATCAAAATGCTTGAGTAT

[1003] SEQ ID NO: 81 - 3xFLAG-UPTAL[mmCentro]-mEGFP-3xmmSTRN3CC Mouse Centromere TAL-mEGFP-3x Mouse Striatin-3 Coiled-Coil Protein Sequence

[1004] MADYKDHDGDYKDHDIDYKDDDDKGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL

[1005] SQHPAALGTVAVTYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGV

[1006] TAMEAVHASRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQA

[1007] LETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETV

[1008] QRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLP

[1009] VLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQ

[1010] AHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGL

[1011] TPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQ

[1012] VVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAI

[1013] ASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRV

[1014] AGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC

[1015] FSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY

[1016] NYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKR DHM VLLEFVTA AGITL GMDEL YKEFGGGGS GGGGSWE VERAELQ ARI AFLQGERKGQENLKKDL VRRIKML EYGGGGSWEVERAELQARIAFLQGERKGQENLKKDLVRRIKMLEYGGGGSWEVERAELQARIAFLQGERKG QENLKKDLVRRIKMLEY

[1017] SEQ ID NO: 82 - 3xFLAG-UPTAL[mrnCentro]-mEGFP-3xmrnSTRN3CC Mouse Centromere TAL-mEGFP-3x Mouse Striatin-3 Coiled-Coil DNA Sequence

[1018] ATGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC AAGGGTACCGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTT

[1019] CGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGC TCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGA GGCGACACACGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTC

[1020] ACGGATGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAA CGTGGCGGCGTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAAC CTGACCCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAAC

[1021] GACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAACAA

[1022] TGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACT

[1023] CCTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTC

[1024] TACCAGTTCTCTGTCAAGCCCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGG

[1025] CAAACAGGCTCTTGAGACCGTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCG

[1026] CAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAG

[1027] TTCTTTGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACA

[1028] GGCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTT

[1029] GTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTG

[1030] TCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTT

[1031] GAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGA

[1032] TTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTTCCAGTTCTTTGTCAAGC

[1033] TCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACC

[1034] GTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTA

[1035] GTCATGACGGTGGCAAACAGGCCCTTGAGACAGTCCAGCGACTCCTCCCAGTTCTCTGTCAAGCCCACGG

[1036] ACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAG

[1037] CGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGA

[1038] CGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACC

[1039] CCAGATCAAGTTGTAGCGATTGCTAGTAACAATGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCC

[1040] TCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGCGG

[1041] TCGACCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAAC

[1042] GACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGATGGATGCAGTGAAAAAGGGATTGCCGC

[1043] ACGCGCCGGAATTGATCCGTAGAGTCAATCGCCGTATTGGCGAACGCACATCCCATCGCGTTGCCGGATC

[1044] CGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAA

[1045] CGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT

[1046] CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAG

[1047] TGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG

[1048] TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG

[1049] GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGC

[1050] ACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCA

[1051] AGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGA

[1052] ACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAG

[1053] CAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTC

[1054] GGCATGGACGAGCTGTACAAGGAATTCGGTGGAGGCGGAAGTGGTGGAGGCGGAAGTTGGGAAGTGGA

[1055] ACGCGCGGAACTGCAGGCGCGCATTGCGTTTCTGCAGGGCGAACGCAAAGGCCAGGAAAACCTGAAAAA

[1056] AGATCTGGTGCGCCGCATTAAAATGCTGGAATATGGTGGTGGGGGTAGCTGGGAGGTCGAGAGAGCCGA

[1057] ATTGCAAGCCCGAATTGCCTTCCTTCAGGGAGAACGCAAGGGGCAAGAGAACTTGAAAAAAGACTTGGT

[1058] CCGCCGCATAAAAATGCTTGAATATGGTGGTGGAGGTTCATGGGAGGTTGAACGTGCCGAACTGCAAGCT

[1059] CGCATTGCGTTCTTACAAGGAGAACGCAAGGGTCAGGAAAACTTAAAAAAGGACCTTGTGCGTCGCATC

[1060] AAAATGCTTGAGTAT

[1061] SEQ ID NO: 83 - 3xmmSgolCC 3x Mouse Shugoshin-1 Coiled-Coil Protein Sequence

[1062] GGGGSGGGGSATLLRYYQDNNRLLVLALENEKSKVREAQDVILQLRKECYYLTCQLGGGGSATLLRYYQDN

[1063] NRLLVLALENEKSKVREAQDVILQLRKECYYLTCQLGGGGSGGGGSATLLRYYQDNNRLLVLALENEKSKVR

[1064] EAQDVILQLRKECYYLTCQL

[1065] SEQ ID NO: 84 - 3xmmSgolCC 3x Mouse Shugoshin-1 Coiled-Coil DNA Sequence

[1066] GGTGGAGGCGGAAGTGGTGGAGGCGGAAGTGCGACCCTGCTGCGCTATTATCAGGATAACAACCGCCTG

[1067] CTGGTGCTGGCGCTGGAAAACGAAAAAAGCAAAGTGCGCGAAGCGCAGGATGTGATTCTGCAGCTGCGC

[1068] AAAGAATGCTATTATCTGACCTGCCAGCTGGGTGGGGGTGGATCAGCTACTCTCCTTCGCTATTACCAAG

[1069] ACAATAATCGCTTGCTGGTACTGGCTTTGGAAAATGAGAAATCCAAGGTGCGAGAAGCACAGGACGTGA

[1070] TACTGCAACTCCGCAAGGAATGTTATTACCTTACCTGCCAACTGGGTGGAGGCGGAAGTGGTGGGGGCGG

[1071] TTCTGCCACCCTGCTGCGTTACTATCAAGATAACAATCGCTTATTGGTATTGGCCCTTGAGAATGAGAAGT

[1072] CCAAGGTTCGTGAAGCGCAGGATGTGATTTTACAACTTCGTAAAGAATGTTACTATCTTACATGCCAACT

[1073] G

[1074] SEQ ID NO: 85 - 3xFLAG-UPTAL[nrniCentro]-mEGFP-3xnmiSgolCC Mouse Centromere TAL-mEGFP-3x Mouse

[1075] Shugoshin-1 Coiled-Coil Protein Sequence

[1076] MADYKDHDGDYKDHDIDYKDDDDKGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL

[1077] SQHPAALGTVAVTYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGV

[1078] TAMEAVHASRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQA

[1079] LETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETV

[1080] QRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLP

[1081] VLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQ

[1082] AHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGL

[1083] TPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAI ASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRV AGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC FSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY NYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKR DHMVLLEFVTAAGITLGMDELYKEFGGGGSGGGGSATLLRYYQDNNRLLVLALENEKSKVREAQDVILQLR KECYYLTCQLGGGGSATLLRYYQDNNRLLVLALENEKSKVREAQDVILQLRKECYYLTCQLGGGGSGGGGS ATLLRYYQDNNRLLVLALENEKSKVREAQDVILQLRKECYYLTCQL

[1084] SEQ ID NO: 86 - 3xFLAG-UPTAL[mmCentro]-mEGFP-3xnmiSgolCC Mouse Centromere TAL-mEGFP-3x Mouse Shugoshin-1 Coiled-Coil DNA Sequence

[1085] ATGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC AAGGGTACCGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTT CGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGC TCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGA GGCGACACACGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTC ACGGATGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAA CGTGGCGGCGTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAAC CTGACCCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAAC GACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAACAA TGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACT CCTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTC TACCAGTTCTCTGTCAAGCCCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGG CAAACAGGCTCTTGAGACCGTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCG CAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAG TTCTTTGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACA GGCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTT GTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTG TCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTT GAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGA TTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTTCCAGTTCTTTGTCAAGC TCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACC GTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTA GTCATGACGGTGGCAAACAGGCCCTTGAGACAGTCCAGCGACTCCTCCCAGTTCTCTGTCAAGCCCACGG ACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAG CGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGA CGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACC CCAGATCAAGTTGTAGCGATTGCTAGTAACAATGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCC TCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGCGG TCGACCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAAC GACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGATGGATGCAGTGAAAAAGGGATTGCCGC ACGCGCCGGAATTGATCCGTAGAGTCAATCGCCGTATTGGCGAACGCACATCCCATCGCGTTGCCGGATC CGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAA CGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAG TGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGC ACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCA AGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGA ACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAG CAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTC GGCATGGACGAGCTGTACAAGGAATTCGGTGGAGGCGGAAGTGGTGGAGGCGGAAGTGCGACCCTGCTG CGCTATTATCAGGATAACAACCGCCTGCTGGTGCTGGCGCTGGAAAACGAAAAAAGCAAAGTGCGCGAA GCGCAGGATGTGATTCTGCAGCTGCGCAAAGAATGCTATTATCTGACCTGCCAGCTGGGTGGGGGTGGAT CAGCTACTCTCCTTCGCTATTACCAAGACAATAATCGCTTGCTGGTACTGGCTTTGGAAAATGAGAAATCC AAGGTGCGAGAAGCACAGGACGTGATACTGCAACTCCGCAAGGAATGTTATTACCTTACCTGCCAACTGG GTGGAGGCGGAAGTGGTGGGGGCGGTTCTGCCACCCTGCTGCGTTACTATCAAGATAACAATCGCTTATT GGTATTGGCCCTTGAGAATGAGAAGTCCAAGGTTCGTGAAGCGCAGGATGTGATTTTACAACTTCGTAAA GAATGTTACTATCTTACATGCCAACTG

[1086] SEQ ID NO: 87 - [G4S] -DogCatcher Protein Sequence GGGGSKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRL IENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQ

[1087] SEQ ID NO: 88 - DogCatcher DNA Sequence GGCGGCGGCGGCAGCAAACTGGGCGAGATTGAATTTATTAAAGTGGACAAAACCGATAAAAAGCCGCTG

[1088] CGTGGTGCCGTGTTTAGCCTGCAGAAACAGCATCCCGACTATCCCGACATCTATGGCGCGATTGATCAGA

[1089] ATGGGACCTATCAAGATGTGCGTACCGGCGAAGATGGTAAACTGACCTTTACGAATCTGAGCGATGGCAA ATATCGCCTGATTGAAAATAGCGAACCCCCGGGCTATAAACCGGTGCAGAATAAGCCGATTGTGAGCTTT CGTATTGTGGATGGCGAAGTGCGTGATGTGACCAGTATTGTGCCGCAG

[1090] SEQ ID NO: 89 - [G4S]-DogTag-[G4S] Protein Sequence

[1091] GGGGSDIPATYEFIDGKHYITNEPIPPKGGGGS

[1092] SEQ ID NO: 90 - DogTag DNA Sequence

[1093] GGCGGCGGCGGCAGCGATATTCCGGCGACCTATGAATTTACCGATGGCAAACATTATATTACCAACGAAC

[1094] CGATTCCGCCGAAAGGCGGCGGCGGCAGC

[1095] SEQ ID NO: 91 - SpyCatcher Protein Sequence

[1096] EQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPD

[1097] GYEVATAITFTVNEQGQVTVNGKATKGDAHI

[1098] SEQ ID NO: 92 - SpyCatcher - DNA Sequence

[1099] GAGCAAGGTCAGTCCGGTGATATGACAATTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTG

[1100] ATGAGGACGGCAAAGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTAC

[1101] ATGGATTTCAGATGGACAAGTGAAAGATTTCTACCTGTATCCAGGAAAATATACATTTGTCGAAACCGCA GCACCAGACGGTTATGAGGTAGCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAA ATGGCAAAGCAACTAAAGGTGACGCTCATATT

[1102] SEQ ID NO: 93 - [G4S]-SpyTag-[G4S] Protein Sequence

[1103] GGGGSAHIVMVDAYKPTKGGGGS

[1104] SEQ ID NO: 94 - SpyTag DNA Sequence

[1105] GGCGGCGGCGGCAGCGCCCACATCGTGATGGTGGACGCCTACAAGCCGACGAAGGGCGGCGGCGGCAGC

[1106] SEQ ID NO: 95 - 3xFLAG-UPTAL[mmCentro]-DogCatcher-SpyTag Mouse Centromere TAL-mEGFP-[G4S]-

[1107] DogCatcher-[G4S]-SpyTag-[G4S] Protein Sequence

[1108] MADYKDHDGDYKDHDIDYKDDDDKGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL

[1109] SQHPAALGTVAVTYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGV

[1110] TAMEAVHASRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQA

[1111] LETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETV

[1112] QRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLP

[1113] VLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQ

[1114] AHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGL

[1115] TPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQ

[1116] VVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAI

[1117] ASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRV

[1118] AGSDQEFGGGGSKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGKLTFTNL

[1119] SDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQSRGGGGSAHIVMVDAYKPTKGGGGS

[1120] SEQ ID NO: 96 - 3xFLAG-UPTAL[mmCentro]-DogCatcher-SpyTag Mouse Centromere TAL-mEGFP-[G4S]-

[1121] DogCatcher-[G4S]-SpyTag-[G4S] DNA Sequence

[1122] ATGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC

[1123] AAGGGTACCGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTT

[1124] CGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGC

[1125] TCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGA

[1126] GGCGACACACGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTC

[1127] ACGGATGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAA

[1128] CGTGGCGGCGTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAAC

[1129] CTGACCCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAAC

[1130] GACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAACAA

[1131] TGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACT

[1132] CCTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTC

[1133] TACCAGTTCTCTGTCAAGCCCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGG

[1134] CAAACAGGCTCTTGAGACCGTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCG

[1135] CAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAG

[1136] TTCTTTGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACA

[1137] GGCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTT

[1138] GTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTG

[1139] TCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTT

[1140] GAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGA

[1141] TTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTTCCAGTTCTTTGTCAAGC TCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACC

[1142] GTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTA

[1143] GTCATGACGGTGGCAAACAGGCCCTTGAGACAGTCCAGCGACTCCTCCCAGTTCTCTGTCAAGCCCACGG

[1144] ACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAG

[1145] CGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGA

[1146] CGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACC

[1147] CCAGATCAAGTTGTAGCGATTGCTAGTAACAATGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCC

[1148] TCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGCGG

[1149] TCGACCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAAC

[1150] GACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGATGGATGCAGTGAAAAAGGGATTGCCGC

[1151] ACGCGCCGGAATTGATCCGTAGAGTCAATCGCCGTATTGGCGAACGCACATCCCATCGCGTTGCCGGATC

[1152] CGATCAAGAATTCGGCGGCGGCGGCAGCAAACTGGGCGAGATTGAATTTATTAAAGTGGACAAAACCGA

[1153] TAAAAAGCCGCTGCGTGGTGCCGTGTTTAGCCTGCAGAAACAGCATCCCGACTATCCCGACATCTATGGC

[1154] GCGATTGATCAGAATGGGACCTATCAAGATGTGCGTACCGGCGAAGATGGTAAACTGACCTTTACGAATC

[1155] TGAGCGATGGCAAATATCGCCTGATTGAAAATAGCGAACCCCCGGGCTATAAACCGGTGCAGAATAAGC

[1156] CGATTGTGAGCTTTCGTATTGTGGATGGCGAAGTGCGTGATGTGACCAGTATTGTGCCGCAGTCTAGAGG

[1157] CGGCGGCGGCAGCGCCCACATCGTGATGGTGGACGCCTACAAGCCGACGAAGGGCGGCGGCGGCAGC

[1158] SEQ ID NO: 97. - 3xFLAG-UPTAL[mmCentro]-DogTag-SpyCatcher

[1159] Mouse Centromere TAL-mEGFP-[G4S]-DogCatcher-[G4S]-SpyTag-[G4S]

[1160] Protein Sequence

[1161] MADYKDHDGDYKDHDIDYKDDDDKGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL

[1162] SQHPAALGTVAVTYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGV

[1163] TAMEAVHASRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQA

[1164] LETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETV

[1165] QRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLP

[1166] VLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQ

[1167] AHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGL

[1168] TPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQ

[1169] VVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAI

[1170] ASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRV

[1171] AGSDQEFGGGGSDIPATYEFTDGKHYITNEPIPPKGGGGSSREQGQSGDMTIEEDSATHIKFSKRDEDGKELAG

[1172] ATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI

[1173] SEQ ID NO: 98 - 3xFLAG-UPTAL[mmCentro]-DogTag-SpyCatcher Mouse Centromere TAL-mEGFP-[G4S]-

[1174] DogCatcher-[G4S]-SpyTag-[G4S] DNA Sequence

[1175] ATGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC

[1176] AAGGGTACCGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTT

[1177] CGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGC

[1178] TCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGA

[1179] GGCGACACACGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTC

[1180] ACGGATGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAA

[1181] CGTGGCGGCGTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAAC

[1182] CTGACCCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAAC

[1183] GACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAACAA

[1184] TGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACT

[1185] CCTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTC

[1186] TACCAGTTCTCTGTCAAGCCCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGG

[1187] CAAACAGGCTCTTGAGACCGTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCG

[1188] CAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAG

[1189] TTCTTTGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACA

[1190] GGCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTT

[1191] GTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTG

[1192] TCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTT

[1193] GAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGA

[1194] TTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTTCCAGTTCTTTGTCAAGC

[1195] TCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACC

[1196] GTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTA

[1197] GTCATGACGGTGGCAAACAGGCCCTTGAGACAGTCCAGCGACTCCTCCCAGTTCTCTGTCAAGCCCACGG

[1198] ACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAG

[1199] CGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGA

[1200] CGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACC

[1201] CCAGATCAAGTTGTAGCGATTGCTAGTAACAATGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCC

[1202] TCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGCGG

[1203] TCGACCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAAC

[1204] GACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGATGGATGCAGTGAAAAAGGGATTGCCGC

[1205] ACGCGCCGGAATTGATCCGTAGAGTCAATCGCCGTATTGGCGAACGCACATCCCATCGCGTTGCCGGATC CGATCAAGAATTCGGCGGCGGCGGCAGCGATATTCCGGCGACCTATGAATTTACCGATGGCAAACATTAT ATTACCAACGAACCGATTCCGCCGAAAGGCGGCGGCGGCAGCTCTAGAGAGCAAGGTCAGTCCGGTGAT ATGACAATTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAGGACGGCAAAGAGTTA GCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGATTTCAGATGGACAAG TGAAAGATTTCTACCTGTATCCAGGAAAATATACATTTGTCGAAACCGCAGCACCAGACGGTTATGAGGT AGCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGCAAAGCAACTAAAGGT GACGCTCATATT

[1206] SEQ ID NO: 99 - UPTAL[hsCentro] Human Centromere Transcription Activator-Like (TAL) Protein Sequence VDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVTYQHIITALPEATHEDI VGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGVTAMEAVHASRNALTGAPLNLTPDQVVA IASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGK QALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALE TVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQR LLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPV LCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLT PDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHL VALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRVA

[1207] SEQ ID NO: 100 - UPTAL[hsCentro] Human Centromere Transcription Activator-Like (TAL) DNA Sequence GTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACA GTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAAC ACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGAGGCGACACA CGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTCACGGATGCG GGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAACGTGGCGGC GTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAACCTGACCCCTG ATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCC AGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAA CAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCAAG TTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTCTACCAGTTCTC TGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCAC TTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCG ATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAG CCCACGGACTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAAC CGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGATTGCT AGTCATGACGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACG GACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCA ACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTAAT ATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTGTCAAGCCCACGGACTAA CTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACT GCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAATGGGGGT GGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAG ATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTCTACC AGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAA CAGGCCCTTGAGACAGTCCAGCGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCAAG TTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTT TGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCAC TCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGC GATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAA GCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTCATGACGGCGGTCGACCGGCGCTGGAGA GCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAACGACCACCTCGTCGCCTTG GCCTGCCTCGGCGGACGTCCTGCGATGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGAATTGATCC GTAGAGTCAATCGCCGTATTGGCGAACGCACATCCCATCGCGTTGCC

[1208] SEQ ID NO: 101 - 3xFLAG-UPTAL[hsCentro]-DogCatcher-SpyTag Human Centromere TAL-mEGFP-[G4S]- DogCatcher-[G4S]-SpyTag-[G4S] Protein Sequence MADYKDHDGDYKDHDIDYKDDDDKGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL SQHPAALGTVAVTYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGV TAMEAVHASRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETV QRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLL PVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLC QAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHG LTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPA QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVA IASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASH

[1209] DGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRVAGS

[1210] DQEFGGGGSKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDG

[1211] KYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQSRGGGGSAHIVMVDAYKPTKGGGGS

[1212] SEQ ID NO: 102 - 3xFLAG-UPTAL[hsCentro]-DogCatcher-SpyTag Human Centromere TAL-mEGFP-[G4S]-

[1213] DogCatcher-[G4S]-SpyTag-[G4S] DNA Sequence

[1214] ATGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC

[1215] AAGGGTACCGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTT

[1216] CGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGC

[1217] TCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGA

[1218] GGCGACACACGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTC

[1219] ACGGATGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAA

[1220] CGTGGCGGCGTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAAC

[1221] CTGACCCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAAC

[1222] GACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTCATGAC

[1223] GGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTC

[1224] CTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTCT

[1225] ACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTAATATTGGTGGC

[1226] AAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATC

[1227] AAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCCAGT

[1228] TCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAG

[1229] GCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTG

[1230] TAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTG

[1231] TCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTT

[1232] GAGACCGTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGA

[1233] TTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTGTCAAGCC

[1234] CACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCG

[1235] TGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGT

[1236] AATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTTCCAGTTCTTTGTCAAGCTCACGGAC

[1237] TCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACG

[1238] CCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTCATGACG

[1239] GTGGCAAACAGGCCCTTGAGACAGTCCAGCGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCC

[1240] TGATCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTT

[1241] CCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCA

[1242] AACAGGCACTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCA

[1243] AGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCCAGTT

[1244] CTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTCATGACGGCGGTCGACCGG

[1245] CGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAACGACCACCTC

[1246] GTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGATGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGG

[1247] AATTGATCCGTAGAGTCAATCGCCGTATTGGCGAACGCACATCCCATCGCGTTGCCGGATCCGATCAAGA

[1248] ATTCGGCGGCGGCGGCAGCAAACTGGGCGAGATTGAATTTATTAAAGTGGACAAAACCGATAAAAAGCC

[1249] GCTGCGTGGTGCCGTGTTTAGCCTGCAGAAACAGCATCCCGACTATCCCGACATCTATGGCGCGATTGAT

[1250] CAGAATGGGACCTATCAAGATGTGCGTACCGGCGAAGATGGTAAACTGACCTTTACGAATCTGAGCGATG

[1251] GCAAATATCGCCTGATTGAAAATAGCGAACCCCCGGGCTATAAACCGGTGCAGAATAAGCCGATTGTGA

[1252] GCTTTCGTATTGTGGATGGCGAAGTGCGTGATGTGACCAGTATTGTGCCGCAGTCTAGAGGCGGCGGCGG CAGCGCCCACATCGTGATGGTGGACGCCTACAAGCCGACGAAGGGCGGCGGCGGCAGC

[1253] SEQ ID NO: 103 - 3xFLAG-UPTAL[hsCentro]-DogTag-SpyCatcher Human Centromere TAL-mEGFP-[G4S]-

[1254] DogCatcher-[G4S]-SpyTag Protein Sequence

[1255] DYKDHDGDYKDHDIDYKDDDDKGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFIHAHIVALSQH

[1256] PAALGTVAVTYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGVTA

[1257] MEAVHASRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALE

[1258] TVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQR

[1259] LLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPV

[1260] LCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQA

[1261] HGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLT

[1262] PAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQV

[1263] VAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIAS

[1264] NGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDG

[1265] GRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRVAGSDQ

[1266] EFGGGGSDIPATYEFTDGKHYITNEPIPPKGGGGSSREQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMEL

[1267] RDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI

[1268] SEQ ID NO: 142 - 3xFLAG-UPTAL[hsCentro]-DogTag-SpyCatcher Human Centromere TAL-mEGFP-[G4S]-

[1269] DogCatcher-[G4S]-SpyTag DNA Sequence GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGGT

[1270] ACCGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCG

[1271] ACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGCTCAGCC

[1272] AACACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGAGGCGAC

[1273] ACACGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTCACGGAT

[1274] GCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAACGTGGC

[1275] GGCGTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAACCTGACC

[1276] CCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGC

[1277] TCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTCATGACGGTGG

[1278] CAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGAT

[1279] CAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTCTACCAG

[1280] TTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACA

[1281] GGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTG

[1282] TAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTG

[1283] TCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTT

[1284] GAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGA

[1285] TTGCTAGTCATGACGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGC

[1286] CCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACC

[1287] GTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTA

[1288] GTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTGTCAAGCCCACGG

[1289] ACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAA

[1290] CGACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAATG

[1291] GGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTTCCAGTTCTTTGTCAAGCTCACGGACTCAC

[1292] CCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTT

[1293] CTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTCATGACGGTG

[1294] GCAAACAGGCCCTTGAGACAGTCCAGCGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGA

[1295] TCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCA

[1296] GTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAAC

[1297] AGGCACTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCAAGT

[1298] TGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTC

[1299] TGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTCATGACGGCGGTCGACCGGCGC

[1300] TGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAACGACCACCTCGTC

[1301] GCCTTGGCCTGCCTCGGCGGACGTCCTGCGATGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGAAT

[1302] TGATCCGTAGAGTCAATCGCCGTATTGGCGAACGCACATCCCATCGCGTTGCCGGATCCGATCAAGAATT

[1303] CGGCGGCGGCGGCAGCGATATTCCGGCGACCTATGAATTTACCGATGGCAAACATTATATTACCAACGAA

[1304] CCGATTCCGCCGAAAGGCGGCGGCGGCAGCTCTAGAGAGCAAGGTCAGTCCGGTGATATGACAATTGAA

[1305] GAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAGGACGGCAAAGAGTTAGCTGGTGCAACTA

[1306] TGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGATTTCAGATGGACAAGTGAAAGATTTCTA

[1307] CCTGTATCCAGGAAAATATACATTTGTCGAAACCGCAGCACCAGACGGTTATGAGGTAGCAACTGCTATT

[1308] ACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGCAAAGCAACTAAAGGTGACGCTCATATT

[1309] SEQ ID NO: 105 - 3xhsSgolCC 3x Human Shugoshin-1 Coiled-Coil Protein Sequence

[1310] GGGGSGGGGSTLLKNYQDNNKMLVLALENEKSKVKEAQDIILQLRKECYYLTCQLGGGGSTLLKNYQDNNK

[1311] MLVLALENEKSKVKEAQDIILQLRKECYYLTCQLGGGGSTLLKNYQDNNKMLVLALENEKSKVKEAQDIILQ

[1312] LRKECYYLTCQL

[1313] SEQ ID NO: 106 - 3xhsSgolCC 3x Human Shugoshin-1 Coiled-Coil DNA Sequence

[1314] GGTGGAGGCGGAAGTGGTGGAGGCGGATCTACACTGCTGAAAAATTACCAAGACAACAACAAAATGTTA

[1315] GTTTTAGCTTTGGAAAATGAAAAATCCAAAGTGAAAGAAGCCCAAGATATCATCCTACAGCTGAGAAAA

[1316] GAATGTTACTATCTCACATGTCAGCTAGGTGGGGGTGGAAGCACCCTGCTGAAGAACTACCAGGACAACA

[1317] ACAAGATGCTGGTGCTTGCCCTGGAGAACGAGAAGAGCAAGGTGAAGGAGGCCCAGGACATCATCCTGC

[1318] AGCTGAGAAAGGAGTGCTACTATCTGACCTGCCAGCTGGGTGGAGGCGGATCTACACTGCTGAAAAATTA

[1319] CCAAGACAACAACAAAATGTTAGTTTTAGCTTTGGAAAATGAAAAATCCAAAGTGAAAGAAGCCCAAGA

[1320] TATCATCCTACAGCTGAGAAAAGAATGTTACTATCTCACATGTCAGCTA

[1321] SEQ ID NO: 107 - 3xFLAG-UPTAL[hsCentro]-mEGFP-3xhsSgolCC Human Centromere TAL-mEGFP-3x Human

[1322] Shugoshin-1 Coiled-Coil Protein Sequence

[1323] MADYKDHDGDYKDHDIDYKDDDDKGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL

[1324] SQHPAALGTVAVTYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGV

[1325] TAMEAVHASRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQA

[1326] LETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETV

[1327] QRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLL

[1328] PVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLC

[1329] QAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHG

[1330] LTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPA

[1331] QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVA

[1332] IASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASH DGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRVAGS

[1333] VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSR

[1334] YPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY

[1335] NSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDH

[1336] MVLLEFVTAAGITLGMDELYKEFGGGGSGGGGSTLLKNYQDNNKMLVLALENEKSKVKEAQDIILQLRKECY

[1337] YLTCQLGGGGSTLLKNYQDNNKMLVLALENEKSKVKEAQDIILQLRKECYYLTCQLGGGGSTLLKNYQDNN KMLVLALENEKSKVKEAQDIILQLRKECYYLTCQL

[1338] SEQ ID NO: 108 - 3xFLAG-UPTAL[hsCentro]-mEGFP-3xhsSgolCC Human Centromere TAL-mEGFP-3x Human

[1339] Shugoshin-1 Coiled-Coil DNA Sequence

[1340] ATGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC

[1341] AAGGGTACCGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTT

[1342] CGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGC

[1343] TCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGA

[1344] GGCGACACACGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTC

[1345] ACGGATGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAA

[1346] CGTGGCGGCGTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAAC CTGACCCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAAC

[1347] GACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTCATGAC

[1348] GGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTC

[1349] CTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTCT

[1350] ACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTAATATTGGTGGC

[1351] AAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATC

[1352] AAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCCAGT

[1353] TCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAG

[1354] GCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTG

[1355] TAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTG

[1356] TCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTT

[1357] GAGACCGTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGA

[1358] TTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTGTCAAGCC

[1359] CACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCG

[1360] TGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGT

[1361] AATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTTCCAGTTCTTTGTCAAGCTCACGGAC

[1362] TCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACG CCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTCATGACG

[1363] GTGGCAAACAGGCCCTTGAGACAGTCCAGCGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCC

[1364] TGATCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTT

[1365] CCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCA

[1366] AACAGGCACTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCA

[1367] AGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCCAGTT

[1368] CTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTCATGACGGCGGTCGACCGG

[1369] CGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAACGACCACCTC

[1370] GTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGATGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGG

[1371] AATTGATCCGTAGAGTCAATCGCCGTATTGGCGAACGCACATCCCATCGCGTTGCCGGATCCGTGAGCAA

[1372] GGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAA

[1373] GTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC

[1374] ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCC

[1375] GCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCG

[1376] CACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCT

[1377] GGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGA

[1378] GTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTT

[1379] CAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCAT

[1380] CGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCC

[1381] AACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACG

[1382] AGCTGTACAAGGAATTCGGTGGAGGCGGAAGTGGTGGAGGCGGATCTACACTGCTGAAAAATTACCAAG

[1383] ACAACAACAAAATGTTAGTTTTAGCTTTGGAAAATGAAAAATCCAAAGTGAAAGAAGCCCAAGATATCA TCCTACAGCTGAGAAAAGAATGTTACTATCTCACATGTCAGCTAGGTGGGGGTGGAAGCACCCTGCTGAA GAACTACCAGGACAACAACAAGATGCTGGTGCTTGCCCTGGAGAACGAGAAGAGCAAGGTGAAGGAGG

[1384] CCCAGGACATCATCCTGCAGCTGAGAAAGGAGTGCTACTATCTGACCTGCCAGCTGGGTGGAGGCGGATC TACACTGCTGAAAAATTACCAAGACAACAACAAAATGTTAGTTTTAGCTTTGGAAAATGAAAAATCCAAA GTGAAAGAAGCCCAAGATATCATCCTACAGCTGAGAAAAGAATGTTACTATCTCACATGTCAGCTA

[1385] SEQ ID NO: 109 - 3xhsSgo2CC 3x Human Shugoshin-2 Coiled-Coil Protein Sequence

[1386] GGGGSGGGGSIFKISLKHNNRALAQALSREKENSRRITTEKMLLQKEVEKLNFENTGGGGSIFKISLKHNNRAL AQALSREKENSRRITTEKMLLQKEVEKLNFENTGGGGSIFKISLKHNNRALAQALSREKENSRRITTEKMLLQK EVEKLNFENT SEQ ID NO: 110 - 3xhsSgo2CC 3x Human Shugoshin-2 Coiled-Coil DNA Sequence

[1387] GGTGGAGGCGGAAGTGGTGGAGGCGGATCTATTTTCAAAATATCTTTAAAGCACAACAACAGGGCATTA GCTCAGGCTCTTAGTAGAGAAAAAGAGAACTCTCGAAGAATTACAACTGAAAAGATGCTATTGCAAAAA GAAGTAGAGAAACTGAATTTTGAGAACACAGGTGGGGGTGGAAGCATCTTCAAGATCAGCCTCAAGCAC AACAACAGGGCCCTGGCCCAGGCCCTGAGCAGAGAGAAGGAGAATAGCAGGAGAATCACCACCGAGAA GATGCTGCTGCAGAAGGAGGTGGAGAAGCTGAATTTCGAGAACACCGGTGGAGGCGGATCGATCTTCAA

[1388] GATCTCCCTGAAGCACAACAACCGCGCCCTGGCCCAGGCCCTGAGCCGCGAGAAGGAGAATAGCCGCCG CATCACCACCGAGAAGATGCTGTTGCAGAAGGAGGTGGAGAAGCTGAATTTCGAGAACACC

[1389] SEQ ID NO: 111 - 3xFLAG-UPTAL[hsCentro]-mEGFP-3xhsSgo2CC Human Centromere TAL-mEGFP-3x Human Shugoshin-2 Coiled-Coil Protein Sequence

[1390] MADYKDHDGDYKDHDIDYKDDDDKGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL SQHPAALGTVAVTYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGV

[1391] TAMEAVHASRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETV QRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLL PVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLC QAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHG

[1392] LTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPA QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVA IASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASH DGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRVAGS VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSR

[1393] YPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY NSHNVYIMADKQKNGIKVNFKIRHNIED GS VQL ADHYQQNTPIGD GP VLLPDNHYL STQSKL SKDPNEKRDH MVLLEFVTAAGITLGMDELYKEFGGGGSGGGGSIFKISLKHNNRALAQALSREKENSRRITTEKMLLQKEVEK LNFENTGGGGSIFKISLKHNNRALAQALSREKENSRRITTEKMLLQKEVEKLNFENTGGGGSIFKISLKHNNRA L AQ AL SREKENSRRITTEKMLLQKE VEKLNFENT

[1394] SEQ ID NO: 112 - 3xFLAG-UPTAL[hsCentro]-mEGFP-3xhsSgo2CC Human Centromere TAL-mEGFP-3x Human Shugoshin-2 Coiled-Coil DNA Sequence

[1395] ATGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC AAGGGTACCGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTT CGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGC

[1396] TCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGA GGCGACACACGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTC ACGGATGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAA CGTGGCGGCGTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAAC CTGACCCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAAC

[1397] GACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTCATGAC GGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTC CTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTCT ACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTAATATTGGTGGC AAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATC

[1398] AAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCCAGT TCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAG GCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTG TAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTG TCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTT

[1399] GAGACCGTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGA TTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTGTCAAGCC CACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCG TGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGT AATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTTCCAGTTCTTTGTCAAGCTCACGGAC

[1400] TCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACG CCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTCATGACG GTGGCAAACAGGCCCTTGAGACAGTCCAGCGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCC TGATCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTT CCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCA

[1401] AACAGGCACTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCA AGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCCAGTT CTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTCATGACGGCGGTCGACCGG CGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAACGACCACCTC GTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGATGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGG

[1402] AATTGATCCGTAGAGTCAATCGCCGTATTGGCGAACGCACATCCCATCGCGTTGCCGGATCCGTGAGCAA GGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAA GTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCC GCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCG CACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCT GGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGA GTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTT CAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCAT

[1403] CGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCC AACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACG AGCTGTACAAGGAATTCGGTGGAGGCGGAAGTGGTGGAGGCGGATCTATTTTCAAAATATCTTTAAAGCA CAACAACAGGGCATTAGCTCAGGCTCTTAGTAGAGAAAAAGAGAACTCTCGAAGAATTACAACTGAAAA GATGCTATTGCAAAAAGAAGTAGAGAAACTGAATTTTGAGAACACAGGTGGGGGTGGAAGCATCTTCAA GATCAGCCTCAAGCACAACAACAGGGCCCTGGCCCAGGCCCTGAGCAGAGAGAAGGAGAATAGCAGGA

[1404] GAATCACCACCGAGAAGATGCTGCTGCAGAAGGAGGTGGAGAAGCTGAATTTCGAGAACACCGGTGGAG GCGGATCGATCTTCAAGATCTCCCTGAAGCACAACAACCGCGCCCTGGCCCAGGCCCTGAGCCGCGAGAA GGAGAATAGCCGCCGCATCACCACCGAGAAGATGCTGTTGCAGAAGGAGGTGGAGAAGCTGAATTTCGA GAACACC

[1405] SEQ ID NO: 113 - 3xhsSTRN3CC 3x Human Striatin-3 Coiled-Coil Protein Sequence

[1406] GGGGSGGGGSWEVERAELQARIAFLQGERKGQENLKKDLVRRIKMLEYGGGGSWEVERAELQARIAFLQGE RKGQENLKKDLVRRIKMLEYGGGGSWEVERAELQARIAFLQGERKGQENLKKDLVRRIKMLEY

[1407] SEQ ID NO: 114 - 3xhsSTRN3CC 3x Human Striatin-3 Coiled-Coil DNA Sequence

[1408] GGTGGAGGCGGAAGTGGTGGAGGCGGATCTTGGGAGGTGGAACGGGCCGAACTGCAGGCCCGGATTGCA TTTCTACAAGGCGAAAGAAAAGGTCAAGAGAACCTGAAGAAGGACTTAGTAAGAAGAATAAAGATGTTA GAGTATGGTGGGGGTGGATCTTGGGAGGTGGAGAGGGCCGAGCTGCAGGCCAGGATCGCCTTCCTCCAG GGCGAGAGAAAAGGCCAGGAGAACCTGAAGAAGGACCTGGTGAGAAGAATCAAGATGCTGGAGTACGG TGGAGGCGGATCTTGGGAGGTGGAGCGCGCCGAGCTGCAGGCCCGCATCGCCTTCCTGCAGGGCGAACG CAAAGGCCAGGAGAACCTGAAGAAGGATTTGGTGCGCCGCATCAAGATGCTGGAGTAC

[1409] SEQ ID NO: 115 - 3xFLAG-UPTAL[hsCentro]-mEGFP-3xhsSTRN3CC Human Centromere TAL-mEGFP-3x Human Striatin-3 Coiled-Coil Protein Sequence

[1410] MADYKDHDGDYKDHDIDYKDDDDKGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL SQHPAALGTVAVTYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGV TAMEAVHASRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETV QRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLL PVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLC

[1411] QAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHG LTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPA QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVA IASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASH DGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRVAGS VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSR

[1412] YPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY NSHNVYIMADKQKNGIKVNFKIRHNIED GS VQL ADHYQQNTPIGD GP VLLPDNHYL STQSKL SKDPNEKRDH MVLLEFVTAAGITLGMDELYKEFGGGGSGGGGSWEVERAELQARIAFLQGERKGQENLKKDLVRRIKMLEY GGGGSWEVERAELQARIAFLQGERKGQENLKKDLVRRIKMLEYGGGGSWEVERAELQARIAFLQGERKGQE NLKKDLVRRIKMLEY

[1413] SEQ ID NO: 116 - 3xFLAG-UPTAL[hsCentro]-mEGFP-3xhsSTRN3CC Human Centromere TAL-mEGFP-3x Human Striatin-3 Coiled-Coil DNA Sequence

[1414] ATGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC AAGGGTACCGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTT CGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGC TCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGA GGCGACACACGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTC

[1415] ACGGATGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAA CGTGGCGGCGTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAAC CTGACCCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAAC GACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTCATGAC GGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTC CTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTCT

[1416] ACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTAATATTGGTGGC AAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATC AAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCCAGT TCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAG GCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTG TAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTG TCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTT GAGACCGTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGA TTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTGTCAAGCC CACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCG TGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGT AATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTTCCAGTTCTTTGTCAAGCTCACGGAC TCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACG CCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCGCAAGTTGTAGCGATTGCTAGTCATGACG GTGGCAAACAGGCCCTTGAGACAGTCCAGCGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCC TGATCAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTT CCAGTTCTTTGTCAAGCTCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCA AACAGGCACTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACTCCTGATCA AGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAACGACTGCTCCCAGTT CTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTCATGACGGCGGTCGACCGG CGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAACGACCACCTC GTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGATGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGG AATTGATCCGTAGAGTCAATCGCCGTATTGGCGAACGCACATCCCATCGCGTTGCCGGATCCGTGAGCAA GGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAA GTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCC GCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCG CACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCT GGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGA GTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTT CAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCAT CGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCC AACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACG AGCTGTACAAGGAATTCGGTGGAGGCGGAAGTGGTGGAGGCGGATCTTGGGAGGTGGAACGGGCCGAAC TGCAGGCCCGGATTGCATTTCTACAAGGCGAAAGAAAAGGTCAAGAGAACCTGAAGAAGGACTTAGTAA GAAGAATAAAGATGTTAGAGTATGGTGGGGGTGGATCTTGGGAGGTGGAGAGGGCCGAGCTGCAGGCCA GGATCGCCTTCCTCCAGGGCGAGAGAAAAGGCCAGGAGAACCTGAAGAAGGACCTGGTGAGAAGAATCA AGATGCTGGAGTACGGTGGAGGCGGATCTTGGGAGGTGGAGCGCGCCGAGCTGCAGGCCCGCATCGCCT TCCTGCAGGGCGAACGCAAAGGCCAGGAGAACCTGAAGAAGGATTTGGTGCGCCGCATCAAGATGCTGG AGTAC

[1417] SEQ ID NO: 117 - 3xFLAG-UPTAL[mmCentro]- mmRec8S / T>D-mEGFP-3xmmSgo2CC

[1418] Mouse Centromere TAL- [mmRec8S / T>D-SCS]-mEGFP-3x Mouse Shugoshin-2 Coiled-Coil Protein Sequence MADYKDHDGDYKDHDIDYKDDDDKGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL SQHPAALGTVAVTYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGV TAMEAVHASRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQA LETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETV QRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLP VLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQ AHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGL TPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAI ASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRV AGSRDPDLDGWLPPELLGLWDHCAQVPQRMLRQRPQLEDEEDVEEERAADEEERRKDEALDEIEVLREAQEP DGPLMLDDELDLEAAEDEKDRDDLIPPEWWAWDEEGQPEPPALPMLPELPEVPLEMPPRPELDDEAVLRAVA SRVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFS RYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN YNSHNVYIMADKQKNGIKVNFKIRHNIED GS VQL ADHYQQNTPIGDGP VLLPDNHYL STQ SKL SKDPNEKRD HMVLLEFVTAAGITLGMDELYKEFGGGGSGGGGSIFKISLKHNNRALARALSKEKENSRRITTEKMQLQKEVE KLNFENTGGGGSIFKISLKHNNRALARALSKEKENSRRITTEKMQLQKEVEKLNFENTGGGGSIFKISLKHNNR AL ARAL SKEKENSRRITIEKMQLQKE VEKLNFENT

[1419] SEQ ID NO: 118 - 3xFLAG-UPTAL[mmCentro]- mmRec8S / T>D-mEGFP-3xnmiSgo2CC

[1420] Mouse Centromere TAL- [mmRec8S / T>D-SCS]-mEGFP-3x Mouse Shugoshin-2 Coiled-Coil DNA Sequence ATGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC AAGGGTACCGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTT CGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGC TCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCACCTATCAGCATATCATCACCGCGTTGCCAGA GGCGACACACGAAGATATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTC ACGGATGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAA CGTGGCGGCGTGACCGCAATGGAGGCAGTGCATGCAAGCCGCAATGCACTGACGGGTGCCCCCCTGAAC CTGACCCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTTGAAACCGTGCAAC GACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTTGTAGCGATTGCTAGTAACAA TGGTGGCAAACAGGCTCTCGAAACCGTACAACGACTCCTCCCAGTTCTCTGTCAAGCCCACGGACTAACT CCTGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGGCAAACAGGCTCTTGAGACCGTCCAACGCCTTC TACCAGTTCTCTGTCAAGCCCACGGACTCACCCCAGATCAAGTTGTAGCGATTGCTAGTCATGACGGTGG CAAACAGGCTCTTGAGACCGTCCAACGCCTTCTACCAGTTCTCTGTCAAGCCCACGGACTAACCCCAGCG CAAGTTGTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAG TTCTTTGTCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACA GGCTCTTGAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGCCTCACCCCGGCGCAAGTT GTAGCGATTGCTAGTAATATTGGTGGCAAACAGGCACTTGAGACGGTTCAGCGCCTCCTTCCAGTTCTCTG TCAAGCCCACGGACTAACTCCTGATCAAGTTGTAGCGATTGCTAGTAATGGGGGTGGCAAACAGGCTCTT GAAACCGTGCAACGACTGCTCCCAGTTCTCTGTCAAGCCCACGGACTCACCCCGGCGCAAGTTGTAGCGA TTGCTAG...

Claims

Claims1. An engineered protein comprising: a) a first domain that is: i) a nucleic acid binding domain capable of directly binding to nucleic acid; or ii) a domain capable of binding to nucleic acid binding protein; and b) at least a second domain that is a separase cleavage substrate domain comprising at least one amino acid sequence that is recognised and bound by a separase, optionally that is cleaved by a separase.

2. The engineered protein of claim 1 wherein the engineered protein does not comprise a domain that is a reporter domain.

3. The engineered protein of claim 1 wherein the engineered protein does not comprise two domains that are reporter domains.

4. The engineered protein of any of claims 2 or 3 wherein the one or more of the reporter domains is a fluorescent protein domain.

5. The engineered protein of any of claims 1-4 wherein the engineered protein does not comprise a domain that is any one or more of: GFP RFP, YFP or mCherry.

6. The engineered protein of any of claims 1-5 wherein the engineered protein does not comprise two or more domains that are fluorescent proteins.

7. The engineered protein of any of claims 1-6 wherein i) the nucleic acid binding domain capable of directly binding to nucleic acid is a DNA binding domain; and / or ii) the domain capable of binding to nucleic acid binding protein is capable of binding to a DNAbinding protein.

8. The engineered protein of any of claims 1-7 wherein the nucleic acid binding domain capable of directly binding to nucleic acid is a DNA binding domain that is a chromatin binding domain.

9. The engineered protein of any of claims 1-8 wherein: i) the nucleic acid binding domain capable of directly binding to nucleic acid is selected from the group comprising or consisting of: a) a leucine-arginine rich domain, optionally Ki67 DNA-binding domain, optionally SEQ ID NO: 1; b) an alpha-solenoid domain, optionally Transcription activator-like [TAL] effector domain optionally SEQ ID NO: 2; c) a basic helix-turn-helix; d) a zinc finger; e) a leucine zipper; f) a winged helix; g) a winged helix-turn-helix; h) helix-loop-helix; i) HMG-box; j) Wor3 domain; k) a histone protein; or l)a centromeric protein binding domain, optionally the centromeric protein binding domain from human Meikin; and / or ii) the domain capable of binding to nucleic acid binding protein is a) a chromo-domain, optionally a Chromobox protein, preferably the human Chromobox protein 5 (CBX5; Heterochromatin Protein 1 alpha) SEQ ID NO: 3; or b) a phosphorylated Histone binding domain.

10. The engineered protein of any of the preceding claims wherein: i) the nucleic acid binding domain capable of directly binding to nucleic acid is selected from the group comprising or consisting of: a) the c-terminal domain of Ki-67, optionally the terminal 326 (amino acids 2930 - 3256) of human Ki-67, optionally has an amino acid sequence of SEQ ID NO.1, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.1; b) the DNA binding alpha-solenoid domain from the TAL01 engineered protein, optionally has an amino acid sequence of SEQ ID NO.2, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.2; or d) the centromeric protein binding domain from human Meikin, optionally has an amino acid sequence of SEQ ID NO.9, or an amino acid sequence with at least 85%,87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.9; and / or ii) the domain capable of binding to nucleic acid binding protein is: a) a methylated histone binding domain from human Chromobox protein 5 (CBX5) optionally has an amino acid sequence of SEQ ID NO.3, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.3; or b) a phosphorylated Histone 2A binding domain from human Shugoshin 2 optionally has an amino acid sequence of SEQ ID NO.4, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.4.

11. The engineered protein of any of the preceding claims wherein the amino acid sequence that is recognised and bound, by separase, optionally is cleavable by separase, is selected from the group comprising or consisting of: a) an amino acid sequence cleavable by human separase; b) the separase binding site from human Securin of SEQ ID NO.4 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.4; c) the separase cleavage site from human Meikin of SEQ ID NO.5 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.5; d) the separase cleavage site from human Rec8 of SEQ ID NO.6 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.6; e) the separase cleavage site derived from human Rec8 containing substitutions of serine and / or threonine residues for bulky amino acids, preferably aspartate, mimicking phosphorylated threonines and / or serines of SEQ ID NO.8 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.8; and / or f) an amino acid sequence cleavable by separase belonging to human, murine, other rodent, equine, bovine, porcine, ovine, canine, feline, marsupial, camelid, non-human primate, northern white rhinoceros, southern white rhinoceros, African elephant, Asian elephant, endangered or threatened mammalian animal species.

12. The engineered protein of any of claims 1-7 wherein the second domain that is a separase cleavage substrate domain comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid sequences capable of being recognised and bound by, or cleaved by, a separase.

13. The engineered protein of any of the preceding claims wherein the engineered protein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs.

14. The engineered protein of any of the preceding claims wherein the second domain that is a separase cleavage substrate domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs.

15. The engineered protein of any of claims 13 or 14 wherein the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs are positioned appropriately relative to the separase cleavage site, such that separase binds to the engineered protein and cleaves the cleavage site, optionally between 50 to 125 amino acids C-terminally relative to the separase cleavage site.

16. The engineered protein of any of the claims 13-15 wherein the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs are arranged in tandem.

17. The engineered protein of any of the preceding claims wherein the second domain comprises a sequence of LPELPE [SEQ ID NO: 124] and / or LPELPELPE [SEQ ID NO: 125].

18. The engineered protein of any of the preceding claims wherein the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs are not arranged directly in tandem, optionally have a spacer sequence between the LPE motifs.

19. The engineered protein of any of the preceding claims wherein the second domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs and also comprises one or more separase cleavage motifs, optionally with an ExxR consensus where x denotes any amino acid.

20. The engineered protein of any of the preceding claims wherein the second domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs and also comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more separase cleavage motifs optionally wherein each separase cleavage motif comprises an ExxR motif.

21. The engineered protein of any of the preceding claims wherein the second domain of the engineered protein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LPE motifs and does not comprise one or more separase cleavage motifs for example with an ExxR consensus.

22. The engineered protein of any of the preceding claims wherein the second domain of the engineered protein, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more separase cleavage motifs, optionally with an ExxR consensus, and does not comprise one or more LPE motifs.

23. The engineered protein of any of the preceding claims wherein the second domain of the engineered protein comprises two separase cleavage motifs and a tandem LPE motif of sequence LPELPE.

24. The engineered protein of any of the preceding claims wherein the second domain comprises two or more separase cleavage motifs and the two or more separase cleavage motifs have the same sequence.

25. The engineered protein of any of claims 12-24 wherein at least two of the amino acid sequences that are capable of being recognised and bound by, or cleaved by, a separase have different amino acid sequences.

26. The engineered protein of any of claims 12-24 wherein all of the amino acid sequences capable of being recognised and bound by, or cleaved by, a separase are the same amino acid sequence.

27. The engineered protein of any of the preceding claims wherein the second domain comprises a first separase cleavage motif that has a sequence of SEQ ID NO: 126 and a second separase cleavage motif that has a sequence of SEQ ID NO: 127.

28. The engineered protein of any of the preceding claims the second domain comprises a sequence of SEQ ID NO: 126, a sequence of SEQ ID NO: 127, and a sequence of SEQ ID NO: 124 (LPELPE).

29. The engineered protein of any of the preceding claims wherein the second domain comprises an engineered Rec8 protein domain that comprises one or moresubstitutions of a non-negatively charged amino acid to a negatively charged amino acid.

30. The engineered protein of any of claim 29 wherein the negatively charged amino acid is aspartic acid.

31. The engineered protein of any of claims 29 or 30 wherein the Rec8 domain is from a human or a mouse.

32. The engineered protein of any of claims 29-31 wherein the Rec8 domain comprises a sequence that comprises or consists of SEQ ID NO: 51 or 7 but where one or more residues has been substituted to a negative amino acid, for example to an aspartic acid, relative to the sequence of SEQ ID NO: 51 or 7, or comprises or consists of a sequence with at least 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or 100% sequence identify to SEQ ID NO: 51 or 7 but where one or more residues has been substituted to a negative amino acid, for example to an aspartic acid, relative to the sequence of SEQ ID NO: 51 or 7.

33. The engineered protein of any of claims 29-32 wherein one or more serine and / or threonine residues of SEQ ID NO: 51 or 7 are substituted with a negative amino acid, for example aspartic acid.

34. The engineered protein of any of claims 29-33 wherein the engineered Rec8 domain has a sequence that comprises or consists of SEQ ID NO: 59 or 120 or comprises or consists of a sequence with at least 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or 100% sequence identify to SEQ ID NO: 59 but residues that are an aspartic acid in SEQ ID NO: 59 are retained as an aspartic acid.

35. The engineered protein of any of claims 29-34 wherein the engineered Rec8 domain comprises or consists of a sequence of SEQ ID NO: 59 or 120.

36. The engineered protein of any of claims 29-35 wherein the engineered Rec8 domain comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more negatively charged residues.

37. The engineered protein of any of the preceding claims wherein the second domain comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 or more negatively charged residues.

38. The engineered protein according to any of the preceding claims wherein when the engineered protein is present in oocytes, optionally when the engineered protein is administered to an oocyte, optionally via microinjection: a) the proportion of chromosome premature separated sister chromatids (PSSC) decreases, optionally significantly decreases; b) wherein the amount of cohesin does not decrease, or does not significantly decrease, measurable by quantitative or semi-quantitative laboratory methods; c) the proportion of chromosome non-disjunction increases, optionally significantly increases in eggs matured from treated oocytes compared to untreated oocytes; d) the proportion of chromosome non-disjunction increases, optionally significantly increases in oocytes from females genetically predisposed for subfertility or infertility; and / or e) the proportion of chromosome non-disjunction increases or significantly increases, in oocytes modified by pharmacological substances or introduction of proteins causing the oocytes to be predisposed to chromosome instability.

39. The engineered protein according to any of the preceding claims wherein the nucleic acid binding domain capable of directly binding to nucleic acid is selected from the group comprising or consisting of: the Ki67 DNA-binding domain, optionally SEQ ID NO: 1 or the Transcription activator-like [TAL] effector domain optionally SEQ ID NO: 2, or the domain capable of binding to nucleic acid binding protein is the chromobox protein optionally SEQ ID NO.3, or the phosphorylated Histone 2A binding domain from human Shugoshin 2 optionally SEQ ID NO.4, optionally an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.1 or 2 or 3 or 4; and the amino acid sequence cleavable by a separase is the binding site from human securing, optionally SEQ ID NO.5, or is the separase cleavage site from human Meikin, optionally SEQ ID NO.6, or is the separase cleavage site from human Rec8, optionally SEQ ID NO.7, or is the separase cleavage site derived from human Rec8 containing substitutions of serine and threonine residues for aspartate, optionally SEQ ID NO.8 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.5 or 6 or 7 or 8.

40. The engineered protein of any one of the preceding claims, wherein the engineered protein comprises a further domain that comprises at least one amino acid sequence that recruits at least one phosphatase activity.

41. The engineered protein according to claim 40 wherein the further domain comprises at least two or more amino acid sequences that recruit at least one phosphatase activity, optionally wherein at least two or more of the amino acid sequences that recruit at least one phosphatase activity are different amino acid sequences and recruit different phosphatases or wherein at least two or more of the amino acid sequences that recruit at least one phosphatase activity are the same amino acid sequences and recruit multiple molecules of the same phosphatase.

42. The engineered protein according to any of claims 40 or 41 wherein the amino acid sequence that recruits a phosphatase activity comprises or consists of any one or more of: a) the human Shugoshin 1 PP2A interaction peptide of SEQ ID NO.10 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.10; the PP2A subunit A; b) the human Shugoshin 2 PP2A interaction peptide of SEQ ID NO.11 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.11; c) the human Striatin 3 PP2A interaction peptide of SEQ ID NO.12 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.12; and / or c) the human PP2A scaffold subunit A (PPP2A alpha) protein of SEQ ID NO.13 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.13; e) the human PP2A regulatory subunit B' (PPP2R5C) protein of SEQ ID NO.14 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.14; and / or, f) the human PP2A catalytic subunit C (PPP2C alpha) protein of SEQ ID NO.15 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.15.

43. The engineered protein according to any of claims 40-42 wherein the further domain recruits a phosphatase activity when the engineered protein is expressed in a host cell, optionally wherein the host cell is a mammalian cell, optionally a human cell, a murine cell; a rodent cell; an equine cell; a bovine cell, a porcine cell, an ovine cell, a canine cell, a feline cell, a marsupial cell, a camelid cell, a cell of a non-human primate, a cell of a northern white rhinoceros, a cell of a southern white rhinoceros, a cell of an African elephant, a cell of an Asian elephant, a cell of an endangered or threatened mammalian animal species.

44. The engineered protein according to any of claims 40-42 wherein the phosphatase activity recruited by the second domain is a PP2A complex phosphatase activity.

45. The engineered protein according to any of claims 40-44 wherein when expressed in a target cell the engineered protein is able to simultaneously bind to chromatin and recruit phosphatase activity.

46. The engineered protein according to any of claims 40-45 wherein when expressed in a host cell: a) the proportion of phosphorylated cohesin is decreased, optionally significantly decreased, measurable by determining a decrease in the phosphorylated status of endogenous proteins in regions located at or near to the engineered protein, and / or a decrease in chromosome premature separated sister chromatids (PSSC); b) the amount of cohesin does not decrease, optionally does not significantly decrease, measurable by quantitative or semi-quantitative laboratory methods; c) the proportion of chromosome non-disjunction increases, optionally significantly increases, in eggs matured from treated oocytes compared to untreated oocytes from females advanced in maternal age; d) the proportion of chromosome non-disjunction increases, optionally significantly increases, in oocytes from females genetically predisposed for subfertility or infertility; and / or e) the proportion of chromosome non-disjunction increases, optionally significantly increases in oocytes modified by pharmacological substances or introduction of proteins causing the oocytes to be predisposed to chromosome instability.

48. An engineered Rec8 protein domain that comprises one or more substitutions of a non-negatively charged amino acid to a negatively charged amino acid.

49. The engineered Rec8 protein domain of claim 48 wherein the negatively charged amino acid is aspartic acid.

50. The engineered Rec8 protein domain of any of claims 48 or 49 wherein the Rec8 domain is from a human or a mouse.

51. The engineered Rec8 protein domain of any of claims 48-50 wherein the domain comprises both a LPE or LPELPE sequence, and one or two separase cleavage motifs.

52. The engineered Rec8 protein domain of any of claims 48-51 wherein the engineered Rec8 domain has a sequence that comprises or consists of SEQ ID NO: 51 or 7 but where one or more residues has been substituted to a negative amino acid, for example to an aspartic acid, relative to the sequence of SEQ ID NO: 51 or 7, or comprises or consists of a sequence with at least 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or 100% sequence identify to SEQ ID NO: 51 or 7 but where one or more residues has been substituted to a negative amino acid, for example to an aspartic acid, relative to the sequence of SEQ ID NO: 51 or 7.

53. The engineered Rec8 protein domain of any of claims 48-52 wherein one or more serine and / or threonine residues of SEQ ID NO: 51 or 7 are substituted with a negative amino acid, for example aspartic acid.

54. The engineered Rec8 protein domain of any of claims 48-53 wherein the engineered Rec8 domain has a sequence that comprises or consists of SEQ ID NO: 59 or 120 or comprises or consists of a sequence with at least 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or 100% sequence identify to SEQ ID NO: 59 but residues that are an aspartic acid in SEQ ID NO: 59 are retained as an aspartic acid.

55. The engineered Rec8 protein domain of any of claims 48-54 wherein the engineered Rec8 domain comprises or consists of a sequence of SEQ ID NO: 59 or 120.

56. The engineered Rec8 protein domain of any of claims 48-55 wherein the engineered Rec8 domain comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more negatively charged residues.

57. An engineered protein comprising at least: a) a first domain that is: i) a nucleic acid binding domain capable of directly binding to nucleic acid, or ii) a domain capable of binding to nucleic acid binding protein; and b) a second domain that comprises at least one amino acid sequence that recruits at least one phosphatase activity.

58. The engineered protein of claim 57 wherein the nucleic acid binding domain is a DNA binding domain.

59. The engineered protein of claim 58 wherein the DNA binding domain is a chromatin binding domain.

60. The engineered protein of claim 58 or 59 wherein i) the nucleic acid binding domain capable of directly binding to nucleic acid is selected from the group comprising or consisting of: a) a leucine-arginine rich domain, optionally Ki67 DNA-binding domain, optionally SEQ ID NO: 1; b) an alpha-solenoid domain, optionally Transcription activator-like [TAL] effector domain optionally SEQ ID NO: 2; c) a basic helix-turn-helix; d) a zinc finger; e) a leucine zipper; f) a winged helix; g) a winged helix-turn-helix; h) helix-loop-helix; i) HMG-box; j) Wor3 domain; or k) a histone protein; and / or ii) the domain capable of binding to nucleic acid binding protein is selected from the group comprising or consisting of: a) a chromo-domain, optionally a Chromobox protein, preferably the human Chromobox protein 5 (CBX5; Heterochromatin Protein 1 alpha) SEQ ID NO: 3; or b) a phosphorylated Histone binding domain.

61. The engineered protein of any of claims 57-60 wherein i) the nucleic acid binding domain capable of directly binding to nucleic acid is selected from the group comprising or consisting of: a) the DNA binding alpha-solenoid from the TAL01 engineered protein, optionally has an amino acid sequence of SEQ ID NO.2, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.2; or b) an amino acid sequence of SEQ ID NO.2, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.2; c) the centromeric protein binding domain from human Meikin, optionally has an amino acid sequence of SEQ ID NO.9, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.9; and / or ii) the domain capable of binding to nucleic acid binding protein is selected from the group comprising or consisting of: a) the chromobox protein from human CBX5, optionally has an amino acid sequence of SEQ ID NO.3, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.3; b) a phosphorylated Histone 2A binding domain from human Shugoshin 2 optionally has an amino acid sequence of SEQ ID NO.4, or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.4.

62. The engineered protein according to any of claims 57-61 wherein the second domain comprises at least two or more amino acid sequences that recruit at least one phosphatase activity.

63. The engineered protein of claims 62 wherein at least two or more of the amino acid sequences that recruit at least one phosphatase activity are different amino acid sequences and recruit different phosphatases.

64. The engineered protein of claim 62 wherein at least two or more of the amino acid sequences that recruit at least one phosphatase activity are the same amino acid sequences and recruit multiple molecules of the same phosphatase.

65. The engineered protein according to any of claims 57-64 wherein the amino acid sequence that recruits a phosphatase activity comprises or consists of any one or more of: a) the human Shugoshin 1 PP2A interaction peptide of SEQ ID NO.10 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.10; the PP2A subunit A; b) the human Shugoshin 2 PP2A interaction peptide of SEQ ID NO.11 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.11; c) the human Striatin 3 PP2A interaction peptide of SEQ ID NO.12 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.12; and / or c) the human PP2A scaffold subunit A (PPP2A alpha) protein of SEQ ID NO.13 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.13; e) the human PP2A regulatory subunit B' (PPP2R5C) protein of SEQ ID NO.14 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.14; and / or, f) the human PP2A catalytic subunit C (PPP2C alpha) protein of SEQ ID NO.15 or an amino acid sequence with at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.15.

66. The engineered protein according to any of claims 57-65 wherein the second domain recruits a phosphatase activity when the engineered protein is expressed in a host cell, optionally wherein the host cell is a mammalian cell, optionally a human cell, a murine cell; a rodent cell; an equine cell; a bovine cell, a porcine cell, an ovine cell, a canine cell, a feline cell, a marsupial cell, a camelid cell, a cell of a non-human primate, a cell of a northern white rhinoceros, a cell of a southern white rhinoceros, a cell of an African elephant, a cell of an Asian elephant, a cell of an endangered or threatened mammalian animal species.

67. The engineered protein according to any of claims 57-66 wherein the phosphatase activity recruited by the second domain is a PP2A complex phosphatase activity.

68. The engineered protein according to any of claims 57-67 wherein when expressed in a target cell the engineered protein is able to simultaneously bind to chromatin and recruit phosphatase activity.

69. The engineered protein according to any of claims 57-68 wherein when expressed in a host cell: a) the proportion of phosphorylated cohesin is decreased, optionally significantly decreased, measurable by determining a decrease in the phosphorylated status of endogenous proteins in regions located at or near to the engineered protein, and / or a decrease in chromosome premature separated sister chromatids (PSSC); b) the amount of cohesin does not decrease, optionally does not significantly decrease, measurable by quantitative or semi-quantitative laboratory methods relative to untreated control cells; c) the proportion of chromosome non-disjunction increases, optionally significantly increases, in eggs matured from treated oocytes compared to untreated oocytes from females advanced in maternal age; d) the proportion of chromosome non-disjunction increases, optionally significantly increases, in oocytes from females genetically predisposed for subfertility or infertility; and / or e) the proportion of chromosome non-disjunction increases, optionally significantly increases in oocytes modified by pharmacological substances or introduction of proteins causing the oocytes to be predisposed to chromosome instability.

70. A polynucleotide encoding the engineered protein or engineered Rec8 protein domain of any of the preceding claims.

71. The polynucleotide of claim 70 wherein the polynucleotide is DNA.

72. The polynucleotide of claim 70 wherein the polynucleotide is RIMA, optionally mRNA.

73. A composition comprising one or more engineered proteins of any one of the preceding claims, one or more engineered Rec8 domains of any of the preceding claims, or a nucleic acid of any of the preceding claims.

74. A pharmaceutical composition comprising one or more engineered proteins of any one of the preceding claims, one or more engineered Rec8 domains of any of the preceding claims, or a nucleic acid of any of the preceding claims.

75. An oocyte comprising any one or more of the engineered proteins of any of the preceding claims.

76. A method of reducing premature separation of sister chromatids in an oocyte, or reducing aneuploidy, said method comprising administering any one or more of the engineered proteins or polynucleotides or pharmaceutical compositions of any of the preceding claims to the oocyte, optionally before completion of anaphase II, preferably before completion of anaphase I, more preferably before anaphase I, most preferably during or before metaphase I.

77. The method of claim 76 where the method is an in vitro method.

78. The method of claim 76 or 776 wherein said administration is via microinjection into the oocyte, optionally in vitro microinjection, and / or wherein the method comprises a single microinjection in the oocyte of a complex of any of the preceding claims or of a composition comprising a complex of any of the preceding claims or wherein the method comprises at least two separate microinjections of at least two engineered proteins according to the preceding claims, optionally at least two engineered proteins of any of the preceding claims.

79. An engineered protein according to any of the preceding claims, a nucleic acid according to any of the preceding claims or a composition according to any of the preceding claims for use in a method of assisted reproduction, optionally for use in a method of in vitro fertilisation.

80. A method of assisted reproduction, optionally in vitro fertilisation, comprising administering any one or more of the engineered proteins, nucleic acids or compositions of any of the preceding paragraphs to an oocyte

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