Targeted integration in mammalian sequences enhances gene expression

Abstract

Cells with stably integrated exogenous nucleic acid sequences (e.g., transgenes) into their genome are disclosed, said exogenous nucleic acid sequences being located within or near an integration site containing at least a portion of an endogenous retrovirus (ERV) or LTR-retrotransposon (LTR-RT), or replacing sequences containing ERVs or LTR-RTs that are part of or formerly part of the cell genome, as well as methods for producing and using such cells. Advantageously, high levels and / or stable production of transgene expression products can be achieved. Transgene integration and expression can be promoted by modulating cellular DNA repair pathways, for example, by transiently expressing genes encoding proteins that form part of DNA repair pathways during transgene integration.

Description

Background Technology

[0001] The expression of recombinant proteins in mammalian cells is crucial for their biotechnological production and / or therapeutic applications such as gene and cell therapies. The creation of appropriate cell lines requires the successful integration of the transgene into the host genome, followed by expression within those cells. Currently, mainstream strategies for cell line development rely on i) the random integration of the transgene into the cell chromosome, ii) the selection of cells with integrated transgenes, and iii) the selection of specific cells exhibiting optimal productivity characteristics. However, this approach is limited by the copy number of the integrated transgene and the epigenetic effects of the transgene genomic environment, often resulting in low, unstable transcription and / or high clonal variability.

[0002] To overcome these problems commonly associated with cell line development, epigenetic regulators can be used to protect transgenes from negative position effects (Bell and Felsenfeld, 1999). These epigenetic regulators include boundary or insulator elements, locus control regions (LCRs), stabilizing and repressive (STAR) elements, universally active chromatin open elements (UCOEs), and matrix attachment regions (MARs). All of these epigenetic regulators have been used for the production of recombinant proteins in mammalian cell lines (Zahn-Zabal et al., 2001; Kim et al., 2004) and gene therapy (Agarwal et al., 1998; Castilla et al., 1998).

[0003] Publications and other materials used herein to illustrate the invention, and in particular to provide additional details regarding implementation of the invention, including patents and patent applications, are incorporated herein by reference in their entirety. For convenience, these publications are referred to hereinafter by reference to the numbers in the appended bibliography, the names of the authors and the year of publication or the patent / patent publication number.

[0004] Site-specific targeted integration of transgenes is required, particularly for increasing and stabilizing transgene expression in mammalian cells. Site-specific targeted integration of transgenes is also needed because it advantageously results in cells with identical genomic settings, eliminating the need to screen numerous cell clones to identify and select those with high levels of transgene expression. Suitable integration sites are also needed for specific subclasses of transgenes, termed “landing pads.” These integration sites advantageously ensure the stability of the integrated cell line and long-term high expression rates from a single transgene or from low copy numbers. Therefore, there is a particular need in the art to identify and validate suitable insertion sites for transgenes in mammalian cells to achieve efficient and reliable transgene expression. Cell clones for producing therapeutic proteins are also needed, which lack expressed endogenous retroviral sequences (ERVs) and / or do not release viral particles along with the generated therapeutic protein into the cell supernatant or release viral particles along with the generated therapeutic protein into the cell supernatant to a lesser extent. This paper addresses one or more of the above requirements, as well as others. Summary of the Invention

[0005] This discloses the stable integration of exogenous nucleic acid sequences, such as transgenes, into or near an insertion sequence of at least a portion of an endogenous retroviral sequence (ERV) or an LTR-retrotransposon (LTR-RT) in the mammalian genome. In some embodiments, this results in high levels and / or stable production of the transgene expression product. In some embodiments, this is achieved and / or promoted by modulating and / or promoting DNA repair pathways in the host cell.

[0006] An engineered cell, preferably an engineered cell of a mammalian cell line, is disclosed, such as engineered CHO cells including engineered CHO-K1 cells, engineered porcine cells, or engineered human cells, said engineered cell comprising:

[0007] The cell's genome contains:

[0008] At least one locus, said locus comprising an insertion site for an ERV sequence or an insertion site for an LTR-RT sequence, and

[0009] A genetic modification, wherein the genetic modification is integrated into at least one locus.

[0010] The integration of at least one transgene locus and optionally the corresponding allele locus can be an ERV sequence or an LTR-RT sequence insertion locus.

[0011] At least one locus integrating the transgene can be the allelic wild-type (ERV-deleted or LTR-RT-deleted) corresponding locus of an ERV sequence insertion locus or an LTR-RT sequence insertion locus (e.g., an ERV-integrated or LTR-RT-integrated genomic sequence). The transgene can also be integrated near or replace the corresponding ERV or LTR-RT sequence at the insertion locus. The transgene can be integrated into one or two loci, preferably in one or two loci in more than 20%, 30%, or even 40% of the transgene-containing cells in the cell population. The locus can be homozygous and contains at least two copies of, for example, SEQ ID NO:1 or SEQ ID NO:2 or portions thereof. The locus can be heterozygous and contains, for example, both SEQ ID NO:1 and SEQ ID NO:2 or portions thereof.

[0012] Some embodiments involve engineered cells, preferably mammalian cell lines, such as engineered CHO cells including engineered CHO-K1 cells, engineered porcine cells, or engineered human cells, said engineered cells comprising:

[0013] Within the genome of the cell:

[0014] At least one locus, the locus containing an insertion site for an endogenous retroviral (ERV) sequence or an LTR-retrotransposon (LTR-RT) sequence, wherein the at least one locus comprises:

[0015] (i) a) the ERV sequence or the LTR-RT sequence, or b) the insertion site and, optionally, a portion of the sequence in a), and / or

[0016] (ii)(i) allelic wild-type corresponding sequences, and

[0017] At least one transgene encoding at least one transgene expression product, said transgene being integrated into said at least one locus. The cell may be contained on different chromosomes of a CHO cell, such as chromosomes 15 and 9 (i) and (ii). The cell may contain (i) and (ii), and said at least one transgene may be integrated into (ii), but said at least one transgene may not be integrated into (i) or said at least one transgene may also be integrated into (i).

[0018] Some embodiments involve cell populations comprising the engineered cells described herein. The at least one transgene can be integrated into (i) and / or (ii) of more than 20%, 30%, or even more than 40% of the cells in the cell population. The engineered cells of the cell population may comprise (i) and (ii) above. (I) May contain: at least nucleotides 29021 to 40247 (or 29521 to 39747) of SEQ ID NO:1 or a sequence having 95%, 98%, or 99% sequence identity with nucleotides 29021 to 40247 (or 29521 to 39747) of SEQ ID NO:1 and (ii) May contain at least nucleotides 29020 to 31020 (or 29520 to 30520) of SEQ ID NO:2 or a sequence having 95%, 98%, or 99% sequence identity with nucleotides 29020 to 31020 (or 29520 to 30520) of SEQ ID NO:2. In some preferred embodiments, the engineered cells / cell populations lack expressed endogenous retroviral sequences (ERV). In some preferred embodiments, the culture supernatant of the cell population does not contain detectable viral particles.

[0019] The at least one transgenic expression product may be a product of interest, such as a protein of interest. Optionally, the cells / cell population may express the product of interest / protein per unit time at an amount (e.g., picograms / cells / day, μg / L, or mg / L) exceeding the amount of the product of interest / protein when the at least one transgene is integrated into at least one locus in the genome.

[0020] The ERV or LTR-RT can be selected from the group consisting of: type C endogenous retroviral elements (ERV C), MLV (micetic leukemia virus), XMRV (heterophilic murine leukemia virus-associated virus), MMTV (mouse mammary tumor virus), MERV-L (mouse ERV with L-tRNA PBS), VL30 (virus-like 30), IAP (intracisional A-type granules), MusD (Mus D-associated retrovirus), PERV (porcine endogenous retrovirus), KoRV (koala retrovirus), enJSRV (sheep lung adenomavirus), MaLR (mammalian epigenetic LTR retrotransposon), HERV (human endogenous retrovirus) such as HERV-E (human ERV with E-tRNA PBS), HERV-H (human ERV with H-tRNA PBS), HERV-K (human ERV with K-tRNA PBS), HERV-L (human ERV with L-tRNA PBS), etc. Human ERV with PBS), HERV-W (human ERV with W-tRNA PBS), and combinations thereof.

[0021] The ERV sequence or LTR-RT sequence may contain at least one ERV subsequence selected from the group consisting of: gag (group-specific antigen) gene, pol (polymerase) gene, env (enveloping) gene, sequences encoding MA (matrix), CA (capsid), NC (nucleocapsid), sequences encoding SP1 (spacer peptide 1), sequences encoding SP2 (spacer peptide 2), or another domain encoding a protein such as pp12 or p6, being a long terminal repeat (LTR) of the ERV, and combinations thereof, wherein the transgene is optionally integrated into one of the subsequences.

[0022] The cells can be transfected with one or more vectors, said vectors comprising one or more of the genes listed in Table 2 or SEQ ID NO: 25-SEQ ID NO: 28, SEQ ID NO: 38-SEQ ID NO: 58 and / or SEQ ID NO: 59, or with SEQ ID NO: 25-SEQ ID NO: 28, SEQ ID NO: 38-SEQ ID NO: 58 and / or SEQ ID NO: 59. NO: 59 has a sequence with at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity, wherein the cytoplasm of said cells may optionally further contain one or more exogenous chemical inhibitors and / or stimulators of DNA repair pathways (DRP), such as NHEJ inhibitors selected from the group consisting of: NU7441, Olaparib, DNA ligase IV inhibitors, Scr7, KU-0060648, anti-EGFR antibody C225 (cetuximab), compound 401 (2-(4-morpholino)-4H-pyrimido[2,la]isoquinoline-4-one), vanillin, wollamyl, DMNB, IC87361, LY294002, OK-1035, CO 15, NK314, PI 103 hydrochloride and combinations thereof; MMEJ inhibitors selected from the group consisting of: Mirin, Mirin derivatives, PolQ inhibitors, CtIP inhibitors and combinations thereof; HR inhibitors such as RI-1 and BO2; HR stimulants such as RS-1; NHEJ stimulants such as IP6; and any combination of the above inhibitors and / or stimulants. In any of the engineered cells, the locus may have at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence selected from SEQ ID No. 1 and / or SEQ ID No. 2. The transgene may be a landing pad. In some preferred embodiments, the engineered cells are Chinese hamster ovary (CHO) cells, or human cells or porcine cells.

[0023] One embodiment includes a method for integrating a transgene into the genome of a cell, preferably a mammalian cell line, the method comprising:

[0024] (a) Providing at least one transgene as part of a vector (e.g., a plasmid or viral vector) containing the at least one transgene, wherein the vector integrates the transgene into at least one locus of the cell, the locus containing an insertion site for an endogenous retroviral (ERV) sequence or an LTR-retrotransposon (LTR-RT) sequence; or

[0025] (b) Provides at least one transgene, optionally as part of a vector, and at least one nuclease and / or cleavage enzyme, wherein the nuclease and / or cleavage enzyme is preferably encoded by at least one vector, wherein the nuclease and / or cleavage enzyme introduces double-stranded and / or single-stranded breaks into at least one locus of the cell to integrate the transgene therein, the locus containing an insertion site for an endogenous retroviral (ERV) sequence or an LTR-retrotransposon (LTR-RT) sequence, and optionally, provides at least one vector encoding at least one targeting element that guides the at least one nuclease and / or cleavage enzyme;

[0026] Optional upregulation, particularly stimulating at least one primary DNA repair pathway (DRP) in cells, and optional downregulation, particularly stimulating at least one secondary DRP in cells, or vice versa.

[0027] The cells were transfected with at least one of the genetically modified organisms; and

[0028] Optionally, engineered cells containing the transgene integrated into the locus are isolated.

[0029] The cells / cell lines may also be transfected with one or more genes from Table 2 or SEQ ID NO: 25-SEQ ID NO: 28, SEQ ID NO: 38-SEQ ID NO: 58 and / or SEQ ID NO: 59, or sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 25-SEQ ID NO: 28, SEQ ID NO: 38-SEQ ID NO: 58 and / or SEQ ID NO: 59, preferably as part of one or more additional vectors, and / or contacted with chemicals that affect the DNA repair pathway (DRP) of the cells. The cells may also be transfected with one or more additional vectors containing and expressing SEQ ID NO: 25-SEQ ID NO: 28, SEQ ID NO: 38-SEQ ID NO: 58 and / or SEQ ID NO: 59, preferably containing and expressing SEQ ID NO: 25-SEQ ID NO: 28.

[0030] The at least one nuclease and / or cleavage enzyme may be a transposase, integrase, recombinase (e.g., a site-specific recombinase), cleavage enzyme, or nuclease (e.g., a site-specific nuclease), a fusion protein comprising a programmable DNA-binding domain and a nuclease domain, or any combination thereof.

[0031] Homing endonucleases, restriction enzymes, zinc finger nucleases or zinc finger cutting enzymes, meganucleases or meganickases, transcription activator-like effector nucleases or transcription activator-like effector cutting enzymes, RNA-guided nucleases or RNA-guided cutting enzymes, DNA-guided nucleases or DNA-guided cutting enzymes, megaTAL nucleases, BurrH-nucleases, ARCUS nucleases, their modified or chimeric forms or variants, and combinations thereof, particularly zinc finger nucleases or zinc finger cutting enzymes, transcription activator-like effector nucleases or transcription activator-like effector cutting enzymes, RNA-guided nucleases or RNA-guided cutting enzymes (wherein the RNA-guided nucleases or RNA-guided cutting enzymes are optionally part of the CRISPR system), restriction enzymes, and combinations thereof. The recombinase may be Cre recombinase, FLP recombinase, λ integrase, PhiC31 integrase, Dre recombinase, xb1 integrase, γδ dissociation enzyme, R4 integrase, Tn3 dissociation enzyme, or TP901-1 recombinase. In some embodiments, the nuclease is a transcription activator-like effector nuclease or an RNA-guided nuclease.

[0032] The vectors used in this article can be plasmids or viral vectors, such as AAV vectors.

[0033] The first and / or second DRP can be selected from the group consisting of: excision, typical homology-directed repair (typical HDR), homology recombination (HR), alternative homology-directed repair (Alt-HDR), double-strand break repair (DSBR), single-strand annealing (SSA), synthesis-dependent strand annealing (SDSA), break-induced replication (BIR), alternative end joining (Alt-EJ), microhomology-mediated end joining (MMEJ), DNA synthesis-dependent microhomology-mediated end joining (SD-MMEJ), typical non-homologous end joining repair (C-NHEJ), alternative non-homologous end joining (A-NHEJ), trans-damage DNA synthesis repair (TLS), base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), DNA damage response (DDR), blunt end joining, single-strand break repair (SSBR), interstrand crosslinking repair (ICL), Fanconi anemia (FA) pathway, and combinations thereof.

[0034] In some embodiments, the at least one first DRP is homologous recombination (HR) and the at least one second DRP is one or more non-homologous end-joining (NHEJ) DNA repair pathways; the at least one first DRP may be an Alt-EJ pathway such as MMEJ, and the at least one second DRP may be one or more non-homologous end-joining (NHEJ) DNA repair pathways; the at least one first DRP may be an Alt-EJ pathway such as MMEJ, and the at least one second DRP may be a homologous recombination (HR) DNA repair pathway; or

[0035] The at least one first DRP can be an Alt-EJ pathway such as MMEJ, and the at least one second DRP can be one or more alternative DNA repair pathways.

[0036] Interference / alteration of DRP can be the same upregulation and can take the form of: a) expressing at least one component of the DRP in the cells, including causing overexpression of at least one component of the DRP, b) introducing at least one component of the DRP into the cells, and / or c) contacting the cells with at least one stimulator of the component of the DRP, such as a chemical stimulator, such as an HR stimulator like RS-1, and / or an NHEJ stimulator like IP6.

[0037] Interference / modification can be downregulation and can take the following forms: a) contacting the cells with at least one inhibitor of a component of the DRP, said inhibitor being, for example, a chemical inhibitor, such as an NHEJ inhibitor selected from the group consisting of: NU7441, olaparib, DNA ligase IV inhibitor, Scr7, KU-0060648, anti-EGFR antibody C225 (cetuximab), compound 401 (2-(4-morpholino)-4H-pyrimidino[2,la]isoquinoline-4-one), vanillin, wollamyl, DMNB, IC87361, LY294002, OK-1035, CO 15, NK314, PI 103 hydrochloride and combinations thereof; MMEJ inhibitors selected from the group consisting of: Mirin, Mirin derivatives, PolQ inhibitors, CtIP inhibitors and combinations thereof; HR inhibitors such as RI-1 and BO2,

[0038] b) By contacting or expressing at least one inhibitory nucleic acid, such as miRNA, siRNA, or shRNA, in the cells to inactivate or downregulate at least one component of DRP, and / or

[0039] c) Expressing a protein in the cells that inhibits the DRP or any combination thereof.

[0040] One implementation includes engineered cells produced by one of the methods disclosed herein.

[0041] Another embodiment includes a kit for introducing at least one transgene into cells, said kit comprising:

[0042] In a container: a vector encoding a nuclease and / or cleavage enzyme targeting at least one locus, said locus comprising an insertion site of an endogenous retroviral (ERV) sequence or an LTR-retrotransposon (LTR-RT) sequence, such as SEQ ID NO: 1 and SEQ ID NO: 2, preferably comprising (i) nucleotides 29021 to 40247 of SEQ ID NO: 1 or a sequence having 95%, 98%, or 99% sequence identity with nucleotides 29021 to 40247 of SEQ ID NO: 1 and (ii) at least nucleotides 29020 to 31020 of SEQ ID NO: 2 or a sequence having 95%, 98%, or 99% sequence identity with nucleotides 29020 to 31020 of SEQ ID NO: 2, or a locus comprising nucleotides 29521 to 39747 of SEQ ID NO: 1 or a sequence having 95%, 98%, or 99% sequence identity with nucleotides 29020 to 31020 of SEQ ID NO: 2, or a locus comprising nucleotides 29521 to 39747 of SEQ ID NO: 1 or a sequence having 95%, 98%, or 99% sequence identity with nucleotides 29021 ... (ii) a sequence having 95%, 98%, or 99% sequence identity of nucleotides 29521 to 4247 of SEQ ID NO:1 and (ii) a locus comprising nucleotides 29520 to 30520 of SEQ ID NO:2 or a sequence having 95%, 98%, or 99% sequence identity with nucleotides 29520 to 30520 of SEQ ID NO:2, including an ERV sequence or LTR-RT sequence integrated into the insertion site, such as SEQ ID NO:3; and optionally at least one vector encoding at least one targeting element that guides the at least one nuclease and / or cleavage enzyme.

[0043] Optionally, at least one carrier in a separate container encodes at least one targeting element that guides the at least one nuclease and / or cleavage enzyme.

[0044] At least one stimulator and / or inhibitor of the DNA repair pathway (DRP) in a separate container, and / or

[0045] One or more vectors comprising one or more genes encoding one or more of the DRP proteins listed in Table 2 or SEQ ID NO: 25-SEQ ID NO: 28, SEQ ID NO: 38-SEQ ID NO: 58 and / or SEQ ID NO: 59, or sequences having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO: 25-SEQ ID NO: 28, SEQ ID NO: 38-SEQ ID NO: 58 and / or SEQ ID NO: 59;

[0046] Instructions for use of the at least one nuclease and / or cleavage enzyme and the at least one stimulator and / or inhibitor to transfect the cells with the at least one transgene. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of a locus with an integration site, showing an integration site containing an ERV sequence (here, an allele containing ERVC 109F (SEQ ID NO: 3)) and its wild-type corresponding allele.

[0048] Figure 2A and Figure 2B This demonstrates a dual-CRISPR-based approach for locating the ERV C109F gene at the wild-type allele ( Figure 2A ) and ERV C 109F allele ( Figure 2B Targeted genetic integration in ).

[0049] Figure 3A and Figure 3B Demonstrates the ability to target integration into the ERV C 109F allele ( Figure 3A ) or wild-type allele ( Figure 3B The vector in the targeted integration uses a vector without homologous sequences, whether or not the Alt-EJ repair pathway is stimulated.

[0050] Figure 4A and Figure 4B Demonstrates the ability to target integration into the ERV C 109F allele ( Figure 4A ) or wild-type allele ( Figure 4B The targeted integration uses a vector with a homologous sequence, whether or not the HR repair pathway is stimulated.

[0051] Figure 5A and Figure 5B Showing the effect of targeting the wild-type allele ( Figure 5A ) and ERV C 109F allele ( Figure 5B The development of TaqMan qPCR analysis. The thick horizontal line in the box indicates the location of the amplicon in the TaqMan qPCR analysis.

[0052] Figure 6 The percentage of clones containing transgene integration in two alleles, in the WT allele, in the ERV C109F allele, or in a random locus, with or without DNA homologous sequences and with or without DPR stimulation, is shown.

[0053] Figure 7A , Figure 7B and Figure 7CFluorescence in situ hybridization using transgenic probes to detect integration events at the ERV locus is shown. Notably, due to chromosomal rearrangement, an "ERV locus" can be found on two different chromosomes (chromosomes 15 and 9) in CHO cells.

[0054] Figure 8A , Figure 8B , Figure 8C and Figure 8D The GFP fluorescence measurements for each type of cell clone are shown: no DNA homology / with Alt-EJ stimulation ( Figure 8A ), No DNA homology / No Alt-EJ stimulation ( Figure 8B ), has DNA homology / has HR stimulation ( Figure 8C ) and those with DNA homology / without HR stimulation ( Figure 8D )(and Figure 6 compared to).

[0055] Figure 9 This is a comparison of GFP fluorescence results obtained using clones that target integration with DNA sequence homology on the transgenic vector, comparing regulation by HR mechanism with and without HR mechanism regulation.

[0056] Figure 10A , Figure 10B and Figure 10C It is the integration site of the C-type ERV 109F sequence on chromosome 15. Figure 10A ) and its wild-type allele corresponding locus on chromosome 9 ( Figure 10B A schematic diagram. The top image shows a triangle below the DNA sequence or located at the bottom. Figure 10C The boxes in the diagram represent CRISPR cleavage sites.

[0057] Figure 11A and Figure 11B The vector used for transfection to obtain CHO cell clones producing trastuzumab is shown. On the left... Figure 11A The image shows the co-transfection vector used for transient expression. Figure 11B The image shows immunoglobulin (Ig) expression vectors, namely vectors carrying the Tras_Hc (heavy chain) and Tras_Lc (light chain) sequences, and their integration sites on chromosomes 15 and 9, respectively.

[0058] Figure 12A , Figure 12B , Figure 12C and Figure 12D This is a schematic diagram of four types of clones characterized after the transgene was targeted and integrated into the ERV109F genomic locus in CHO-M cells. Figure 12AThe transgene integrates at two alleles at the locus (referred to as "integrated at two loci" or simply "two locations" in the diagram) (the ERV109F allele on chromosome 15 and the wild-type allele on chromosome 9). Figure 12B The transgene integrated at the ERV109F allele on chromosome 15, but not at the wild-type allele on chromosome 9 (referred to as "integration at the ERV locus" or simply "ERV" in the diagram). Figure 12C The transgene integrated at the wild-type allele on chromosome 9, but not at the ERV109F allele on chromosome 15 (referred to in the figure as "integration at the locus without ERV" or simply "ERV deletion"). Figure 12D The transgene did not integrate into any allele at the locus, but rather randomly integrated into the host chromosome. The gray arrows represent PCR primers used to characterize the integration site of the transgene genome in the clone.

[0059] Figure 13A , Figure 13B and Figure 13C The fold decrease in ERV C109F expression after transgene integration is shown compared to parental CHO-M cells. Total RNA was extracted from the specified cell clones, and viral RNA levels were determined by RT-qPCR and processed using the Delta Delta Ct (cycle threshold) method. Shading corresponds to... Figures 12A-12C The shadow shown. Figure 13A Low-titer cultures were described. Figure 13B The medium-titer cultures were described, and Figure 13C High-titer cultures were described.

[0060] Figure 14A , Figure 14B and Figure 14C The assay shows the levels of trastuzumab production in the supernatant of various CHO cell clone types. For clone types that have integrated the Tras expression construct at the designated genomic locus (allele), ELISA was performed on cell-free supernatant from 3-day cultures in 96-well plates. The figure shows the titers of trastuzumab antibodies obtained from the cultures, indicating low ( Figure 14A ),medium( Figure 14B ) and high ( Figure 14C The level of production.

[0061] Figures 15A-15F Measurements of trastuzumab productivity of clones cultured in 3 ml of medium with 300,000 cells / ml of starting material during a 10-day fed-batch culture in 24-well plates are provided. Figure 15A(low titer) Figure 15B (medium titer) and Figure 15C (High titer), or a measure of trastuzumab productivity in clones cultured in a 96-well plate. Figure 15D (low titer) Figure 15E (medium titer) and Figure 15F (High titer)). Using LabChip LCGXII (Perkin Elmer, Inc.) performed Tras protein titer measurements. The shading corresponds to the name... Figures 13A-13C and Figures 14A-14C The shaded areas shown in each diagram from left to right represent: Transgenic integration at both alleles at the locus ("both") (ERV109F allele on chromosome 15 and wild-type allele on chromosome 9); Transgenic integration at the ERV109F allele on chromosome 15 but not at the wild-type allele on chromosome 9 ("ERV"); Transgenic integration at neither allele at the locus but randomly integrated into the host chromosome; Transgenic integration at the wild-type allele on chromosome 9 but not at the ERV109F allele on chromosome 15 ("ERV deletion").

[0062] Figure 16A and Figure 16B Depicting in 15 Automated microbioreactor system (SARTORIUSStedim, Germany) for the processing of... Figure 15C ( Figure 16A )and Figure 15F ( Figure 16B Four high-yielding clones were tested for 14 days to assess their productivity on a larger scale. Detailed Implementation

[0063] This document discloses cells and cell lines for generating transgenes, as well as methods for preparing and using them. To generate the transgene of interest, one or more ERV or LTR-RT loci in the cell genome are targeted for transgene integration and expression. In some embodiments, the ERV sequence capable of forming viral particles may be eliminated, or at least made or has been made non-functional in terms of viral particle production. The targeting locus of ERV or LTR-RT may contain one allele that actually contains the ERV or LTR-RT sequence (or contains the ERV or LTR-RT sequence prior to removal), while the other allele does not contain and is a so-called wild-type allele that has never contained the ERV or LTR-RT sequence. The transgene can be introduced into the cell to preferably integrate at the ERV or LTR-RT locus into an allele containing the ERV or LTR-RT sequence, into an allele not containing the ERV or LTR-RT sequence, or into both an allele containing the ERV or LTR-RT sequence and an allele not containing the ERV or LTR-RT sequence.

[0064] In one instance, the transgene encodes an antibiotic selection gene and a gene encoding a protein of interest (e.g., the heavy and light chains of immunoglobulins or human erythropoietin). The transgene is inserted into a vector containing an upstream promoter and a downstream SGE (Selexis genetic element). A transcription activator-like effector (TALE) cleaving enzyme is engineered to recognize and cleave specific DNA sequences at the ERV locus, 5 bp upstream and 5 bp downstream of the ERV integration site. The selected ERV integrates only at one of two alleles, the other being a so-called wild-type allele that has never contained an ERV. CHO-K1 cells are transfected with a vector carrying the gene encoding the TALE cleaving enzyme, a vector carrying the transgene, and a vector designed for transient expression of MRE11. Cells showing transgene integration into the wild-type allele corresponding to the ERV sequence but not into the ERV-containing allele are selected for production of the protein of interest.

[0065] In another example, a kit is used to create CHO cells that produce the transgene of interest. The CHO cells in this kit have been engineered to remove any integrated ERV sequences that produce viral particles or virus-like particles. The cells have also been engineered to insert a landing pad into the allelic wild-type corresponding locus / allelic region of the ERV sequence. The landing pad encodes green fluorescent protein (GFP). The kit also includes a vector encoding a cleavage enzyme for the sequence in the landing pad and at least one vector encoding at least one targeting element that directs the at least one cleavage enzyme to the landing pad. The kit also includes a vector designed for transient CIRBP expression and a vector into which the transgene of interest can be integrated. After integration of the transgene of interest (single-domain antibody), all vectors are co-transfected into engineered CHO cells. CHO cells that do not express GFP are selected. An expression vector for RS-1, a RAD51 stimulator, is also part of the kit and is added during co-transfection to stimulate homologous recombination (HR).

[0066] The cells / cell populations (the latter often also referred to as cell lines, indicating the homogeneity of cells within a cell population) according to the invention are eukaryotic cells, preferably mammalian cells / cell populations (e.g., human or non-human mammalian cells), capable of being maintained under cell culture conditions. Non-limiting examples of this type of cell are human cells, such as HEK cells (human embryonic kidney), Chinese hamster ovary (CHO) cells, mouse myeloma cells including NSO and Sp2 / O cells, and porcine cells such as LLCPK (porcine kidney epithelial) cells. Modified forms of CHO cells include CHO DG44, CHO-K1, and CHO pro-3. In a preferred embodiment, SURE CHO-M cells are used. TM (SELEXIS SA, Switzerland)

[0067] The insertion site of an endogenous retroviral (ERV) sequence or an LTR-reverse transposon (LTR-RT) sequence in the cell genome is a nucleic acid sequence of no more than 100 nucleotides in length, preferably no more than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, or 2 nucleotides: (i)a) contains an ERV sequence or an LTR-RT sequence, i.e., an ERV sequence or an LTR-RT sequence integrated into the cell genome, which is also referred to herein as an integrated ERV / LTR-RT sequence; (i)b) contains an ERV sequence or an LTR-RT sequence before the complete or partial removal of the ERV sequence or LTR-RT sequence; or (ii) is an allelic wild type, sometimes referred to herein as the ERV-deletion corresponding locus of (i). (i)a) and b) are referred to herein as “ERV sequence or LTR-RT sequence insertion locus / allele” or “ERV or LTR-RT insertion locus / allele”, and (ii) is referred herein as “allele wild-type corresponding locus / allele of ERV sequence insertion locus / allele” or “allele wild-type corresponding locus / allele of LTR-RT sequence insertion locus / allele” or simply “allele wild-type (wt) corresponding sequence” as described above. As will be readily understood by those skilled in the art, loci will exist in which:

[0068] - One allele

[0069] (i)a) Contains an ERV sequence or an LTR-RT sequence, or

[0070] (i)b) The ERV sequence or LTR-RT sequence was included before the complete or partial removal of the ERV sequence or LTR-RT sequence, and / or

[0071] (ii) is the allelic wild-type counterpart of (i).

[0072] A cell that combines at least two distinct alleles of a locus, such as (i)a) and (ii), or (i)b) and (ii), is sometimes referred to herein as a heterozygote of that locus.

[0073] Cells combining (i)a) and (ii) are considered hemizygotes with a single copy of the ERV sequence.

[0074] A cell that has two identical alleles, such as (i)a) and (i)a) at a locus, is called a homozygote at that locus.

[0075] Cells are also within the scope of this invention:

[0076] - The cell contains one allele and a corresponding allele locus, therefore the two alleles (i)a) contain an ERV sequence or an LTR-RT sequence or (i)b) contain an ERV sequence or an LTR-RT sequence prior to the complete or partial removal of the ERV sequence or LTR-RT sequence; and

[0077] - The cell contains one allele and a corresponding allele locus, so the two alleles (ii) are the wild-type alleles corresponding to the ERV sequence or LTR-RT sequence insertion locus.

[0078] A non-limiting example of such insertion sites for ERV sequences is included at the 3' end of SEQ ID NO:1, SEQ ID NO:2, and the 5' end of SEQ ID NO:4. The ERV sequence is shown in SEQ ID NO:3.

[0079] (i) The allelic wild-type corresponding locus, containing corresponding 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, and 2 nucleotides of the integration site, but without any indication of integration of the current or past ERV or LTR-RT sequence. A non-limiting example of such an allelic counterpart of SEQ ID NO: 1 is SEQ ID NO: 2, and the insertion site is the nucleotides surrounding nucleotide 30020. SEQ ID NO: 1 would be “ERV or LTR-RT sequence insertion locus”, while SEQ ID NO: 2 would be the corresponding “allelic wild-type corresponding locus of ERV or LTR-RT sequence insertion locus”.

[0080] A locus is a location on a eukaryotic chromosome where the specific genomic sequence typically comprises up to 60,000 nucleotides of the genome it is located in (see [link to relevant documentation]). Figure 5A and Figure 5B(But not less than 1000, 900, 800, 700, or 600 nucleotides. However, as those skilled in the art know, loci with lengths between 612 and 4,767,747 base pairs can be identified in, for example, the human genome (Taher and Ovcharenko, 2009). Alleles are specific forms of genetic loci, distinguished from other forms by their specific nucleotide sequences. Such loci can also be found on different chromosomes due to these genomic rearrangements in cells that have undergone strong genomic rearrangements and can be maintained under cell culture conditions and used for the production of transgenic products. To capture these configurations of genomic sequences of 1,000 to 60,000 nucleotides that typically define loci, different alleles of such loci may be referred to, for example, as “allelic wild-type counterpart loci of ERV or LTR-RT sequence insertion loci” and “ERV or LTR-RT sequence insertion loci”, as discussed elsewhere herein. An example of such loci is a locus with an ERV-C 109F insertion site. Due to rearrangement, this locus is located on chromosomes 15 and 9 (see also...). Figures 7A-7C On chromosome 15, the ERV-C 109F sequence is integrated into the genome, while the "allele wild-type corresponding locus" is located on chromosome 7. This fact that it is actually a locus on two chromosomes can be inferred from the corresponding 5' and / or 3' sequences surrounding the insertion site (e.g., for ERV-C109F, e.g., nucleotides 1-30020 of SEQ ID NO:1 and SEQ ID NO:2, e.g., nucleotides 1-30020 of SEQ ID NO:2). Therefore, multichromosomal loci have high sequence identity, typically 100%, e.g., for the upstream sequences of the ERV-C 109F insertion site (nucleotides 1-30020 of SEQ ID NO:1 and SEQ ID NO:2). However, slight mismatches may reduce sequence identity to, for example, 99%, 98%, 97%, 96%, or 95%. Furthermore, deletions may be present around the ERV or LTR-RT sequence insertion site.

[0081] Loci containing insertion sites of endogenous retroviral (ERV) sequences or LTR-retrotransposon (LTR-RT) sequences are typically identifiable by sequences of up to 60,000, 50,000, 40,000, or 30,000 nucleotides, but usually less than 20,000 or 10,000 nucleotides, and in some cases less than 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, or 2,000 nucleotides, or by sequences of 1,000 to 600 nucleotides, including approximately 900, 800, or 700 nucleotides. As described above, one such locus is shown in SEQ ID NO:1, and its corresponding wild-type allelic locus is shown in SEQ ID NO:2. As those skilled in the art will understand, within the scope of this invention, integration, such as transgene integration, can occur in any portion of an endogenous retroviral (ERV) sequence (see, for example, SEQ ID NO: 3) or an LTR-retrotransposon (LTR-RT) sequence, or in a chromosomal sequence flanking a locus of the ERV or LTR-RT sequence (ERV flanking sequence or LTR-RT flanking sequence), or in a completely or partially deleted ERV or LTR-RT sequence or ERV or LTR-RT flanking sequence replacing that locus (see, for example, SEQ ID NO: 4 and / or SEQ ID NO: 5). Preferred loci containing an insertion site of an ERV or LTR-RT sequence are loci in which the ERV sequence contains at least a partial, but preferably complete, gag, pol, env, and / or at least one, preferably two, LTRs, and most preferably, all such subsequences of the ERV or corresponding subsequences of the LTR-RT. In some embodiments, the even more preferred locus containing the insertion site of an endogenous retroviral (ERV) sequence or an LTR-retrotransposon (LTR-RT) sequence is the corresponding allelic wild-type locus, which does not contain any ERV or LTR-RT sequence.

[0082] As those skilled in the art will understand, ERV sequences and LTR-RT sequences and their respective loci, other than those shown in SEQ ID NO: 1-SEQ ID NO: 5, are also within the scope of this invention. Some non-limiting examples (such as ERV sequences) are listed in Table 1 as SEQ ID NO: 12-SEQ ID NO: 24.

[0083] Table 1: Common alternative ERVs in CHO cells.

[0084] SEQ ID NO: Examples of alternative ERVs in CHO cells 12 000036F-021-01: 1-25000_Type_C: ERV C 36F 13 000066F-017-01: 1-39146_Type_C: ERV C 66F 14 000132F-023-01: 1-15000_Type_C: ERV C 132F 15 000161F-025-01: 1-17947_Type_C: ERV C 161F 16 000308F-002-01: 1-31438_Type_C: ERV C 308F 17 000312F-023-01: 15000-35000_Type_C: ERV C 312F 18 000322F-036-01: 15000-35000_Type_C: ERV C 322F 19 000351F-017-01: 1-31837Type_C: ERV C 351F 20 000519F-009-01: 1-25000Type_C: ERV C 519F 21 000562F-005-01: 25000-45000_Type_C: ERV C 62F 22 001329F-001-01:1-4677_Type_C:ERV C1329F 23 000386F: 294053-310687_Type_C: expressed ERV type C 368F 24 000506F: 340000-360000_Type_C: expressed ERV type C 506F

[0085] As noted, the locus containing the ERV sequence or LTR-RT sequence and its insertion site have allelic counterparts that also contain the insertion site but may contain the ERV sequence or LTR-RT sequence or may not contain the ERV sequence or LTR-RT sequence. As described above, in some embodiments, no locus may have the ERV sequence or LTR-RT sequence: it may have been engineered or may be engineered to remove the ERV sequence or LTR-RT sequence or portions thereof, and in some embodiments, a portion of the locus, i.e., the sequence flanking the ERV or LTR-RT insertion site / the ERV or LTR-RT sequence inserted therein, is also removed. During transgene integration, the cells may be engineered accordingly, or the transgene may be integrated into cells that have been engineered to remove or alter portions of the ERV sequence or LTR-RT sequence. For example, corresponding alterations and resulting cells are disclosed in U.S. Patent Application 62 / 784,566 and U.S.-designated International Patent Application Publication WO2020 / 136149, the entire contents of which are incorporated herein by reference.

[0086] A hemizygosity for an ERV / LTR-RT sequence refers to the fact that only one copy of a given ERV / LTR-RT sequence exists at a specific locus in a diploid cell. This implies the existence of an "ERV or LTR-RT sequence insertion locus" and its "wild-type allele correspondence at the ERV or LTR-RT sequence insertion locus." Homozygosity for an ERV / LTR-RT sequence means that the alleles at the locus correspond and both carry either an "ERV or LTR-RT sequence insertion" or are both "wild-type alleles" at the specific locus in a diploid cell.

[0087] Transgenic genes can be integrated into loci that are hemizygous or homozygous in terms of the ERV / LTR-RT sequence.

[0088] LTR-reverse transposon (LTR-RT) sequences, also known as mammalian LTR-reverse transposon sequences or MaLR sequences, contain at least two LTR sequences flanking regions encoding two enzymes: at least one gag gene and one pol gene, which can be translated into at least two enzymes, integrase and reverse transcriptase (RT). Unlike ERV, LTR-RT sequences never contain the env gene encoding the envelope protein (ENV) (Havecker et al., 2004).

[0089] An endogenous retroviral (ERV) sequence constitutes the left-over portion of the retrovirus integrated into the cellular genome and contains at least a portion of the gag, pol, and env genes, and / or at least one, preferably two LTRs. Functional units or portions of the ERV sequence are also referred to as ERV subsequences. Therefore, the gag, pol, env, and LTRs are considered ERV subsequences. In a preferred embodiment, at least one, preferably two, of the gag, pol, or env genes express their respective proteins. In an even more preferred embodiment of ERV selection, the ERV sequence releases VP (viral particles) or VLP (virus-like particles). A complete endogenous retrovirus is on average 6kb to 12kb in size and contains the gag, pol, and env genes, which always appear in the same order. Two LTRs (long terminal repeats) flank the coding sequence. Most ERVs are defective because they carry numerous inactivating mutations. Furthermore, they can be inactivated (i.e., not transcribed) by epigenetic silencing effects. However, some ERVs still have open reading frames in their genome and / or they may have transcriptional activity. Mammalian ERVs share strong similarities and likely originate from the genera γ-retroviruses and β-retroviruses, including intracisternal A-particles (IAP or IAPS), feline leukemia virus (FeLV), mouse leukemia virus (MLV), koala epidemic virus (KoRV), and mouse mammary tumor virus (MMTV). ERVs are retained in the genome and may offer certain advantages to cells that integrate them into the genome, including providing a source of genetic diversity and protection against other viral pathogens. However, in the case of transgenic (i.e., protein) expression as described elsewhere in this paper, they can be infectious and pose risks, particularly due to ERV awakening caused by cancer, cellular stress, and / or epigenetic modifications.

[0090] The three main proteins encoded in the retroviral genome are Gag, Pol, and Env. Gag (antigen group), encoded by the gag gene, is a polyprotein that is processed into the matrix and other core proteins, including the nucleoprotein core particle that determines the retroviral core. Pol is a reverse transcriptase, encoded by the pol gene, which has RNase H and integrase functions. Its activity leads to the pre-integration of the viral double-stranded DNA into the host genome via integrase function, and also to reverse transcription after integration into the host genome via RNase function. Env is an envelope protein, encoded by the env gene, located in the viral lipid layer that determines viral orientation.

[0091] The gag gene produces the Gag precursor protein, which is expressed from unspliced ​​viral mRNA. During viral maturation, the Gag precursor protein is cleaved by a viral protease (a product of the pol gene) into four smaller proteins, typically called MA (matrix), CA (capsid), NC (nucleocapsid), and another protein domain (such as pp12 in murine leukemia virus (MLV) or p6 in HIV).

[0092] Viral protease (Pro), integrase (IN), RNase H, and reverse transcriptase (RT) are expressed in the context of the Gag-Pol fusion protein. The Gag-Pol precursor is typically generated by a ribosomal frameshifting event triggered by a specific cis-acting RNA motif (a seven-nucleotide sequence in the distal region of Gag RNA followed by a short stem-loop). When ribosomes encounter this motif, they have approximately 5% of the time to translocate into the pol reading frame without interrupting translation. The frequency of ribosomal frameshifts explains why Gag and Gag-Pol precursors are generated at a ratio of approximately 20:1.

[0093] During viral maturation, the virus-encoded protease cleaves the Pol polypeptide from the Gag and further digests it to separate the protease, RT, RNase H, and integrase activities. These cleavages do not all occur efficiently; for example, approximately 50% of the RT protein remains as a single polypeptide (p65) linked to RNase H (Hope & Trono, 2000).

[0094] The pol gene encodes a reverse transcriptase. During reverse transcription, the polymerase generates a double-stranded DNA copy of the single-stranded genomic RNA dimer present in the viral particle. RNase H removes the original RNA template from the first DNA strand, allowing the synthesis of the complementary DNA strand. The primary functional class of the polymerase is the heterodimer. All pol gene products can be found within the capsid of the released viral particle.

[0095] IN protein mediates the insertion of proviral DNA into the genomic DNA of infected cells. This process is mediated by three different functions of IN.

[0096] Env protein is expressed as a single-splicing mRNA. Initially synthesized in the endoplasmic reticulum, Env migrates through the Golgi complex and undergoes glycosylation there. Env glycosylation is generally essential for infectivity. Cellular proteases cleave the protein into transmembrane and surface domains.

[0097] Viral genomic RNA expressed from some ERVs in the genome can be released from the cell as VPs. Other expressed ERVs may lead to the formation of VLPs, such as RVLPs (retrovirus-like particles), but not VPs, and therefore may not result in the release of particles containing viral genomic RNA. However, those that are typically released are more likely to be infectious.

[0098] In the context of this application, VP refers to a viral particle containing at least a portion of the viral genome. In some cases, a VP may contain full-length viral genomic RNA and thus may be a functional VP. As used in the context of this invention, VLP is a particle that appears to be a VP but lacks any portion of the viral genome.

[0099] The vector according to the invention is a nucleic acid molecule capable of transporting other nucleic acids already linked to it. A plasmid is, for example, one type of vector. A viral vector is another type of vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.

[0100] In some aspects of the invention, vectors are used to transport exogenous nucleic acids into cells or cell populations.

[0101] As used herein, exogenous nucleic acid refers to the nucleic acid introduced into a host cell. The source of exogenous nucleic acid can be, for example, homologous or heterologous nucleic acids expressing, for example, a protein of interest. Accordingly, the term endogenous refers to nucleic acid molecules already present in the host cell. The term heterologous nucleic acid refers to nucleic acid molecules derived from sources other than the host cell species, while homologous nucleic acids refer to nucleic acid molecules derived from the same species as the host cell. Therefore, the exogenous nucleic acid according to the invention can utilize one or both of heterologous and / or homologous nucleic acids.

[0102] For example, the cDNA of the human interferon gene is a heterologous exogenous nucleic acid when introduced into CHO cells, but a homologous exogenous nucleic acid in HeLa cells. When introduced into cells, the exogenous gene can be part of a vector, or it can be introduced without additional endogenous or exogenous nucleic acid sequences.

[0103] A transgene is a foreign nucleic acid that encodes a product such as a protein of interest (also referred to as a "transgene expression product"). In some embodiments, more than one transgene is required to obtain a cell line that produces the product of interest (particularly the protein of interest, such as an antibody). This may require a transgene encoding a light chain and a transgene encoding a heavy chain to produce the antibody, i.e., the protein of interest, as well as an antibiotic-selective transgene for selecting stably transfected cells. The transgene expression product can also be simply a marker protein, such as an antibiotic-selective gene, enhanced green fluorescent protein (GFP), or β-galactosidase (lacZ). In this case, the transgene can be integrated under the control of a specific gene promoter and can replace a completely or partially removed ERV or LTR-RT sequence, or it can be integrated into an allelic wild-type counterpart. Such a transgene can serve as a landing pad for the integration of another transgene, such as a transgene encoding the protein of interest or a transgene expression product that produces the protein of interest (e.g., a therapeutic protein) together with another transgene expression product. For example, the transgene expression product could be the light or heavy chain of an antibody, but the "protein of interest" is an immunoglobulin composed of four chains. However, typically, products of interest (e.g., proteins of interest) are, for example, but not limited to, signaling proteins (e.g., α-IFN, β-IFN, γ-IFN, τ-IFN, ω-IFN), cytokines such as erythropoietin or antibodies such as monoclonal antibodies, proteins of fusion (including fusion proteins), and regulatory RNAs such as siRNA, shRNA, or mRNA. “Protein of interest” refers to therapeutic proteins recovered from cell supernatants and measured therein in picograms / cell / day, μg / L, or mg / L.

[0104] As used herein, transfection refers to the introduction of nucleic acids, including naked or purified nucleic acids or vectors carrying specific nucleic acids, into cells, particularly eukaryotic cells, including mammalian cells. Any known transfection method can be used in the context of this invention. Some of these methods involve enhancing the permeability of biological membranes to carry nucleic acids into cells. Prominent examples are electroporation or microporation. These methods can be used alone or supported by acoustic, electromagnetic, and thermal energy, chemipermeability enhancers, pressure, etc., to selectively increase the flux of nucleic acids into host cells. Other transfection methods are also within the scope of this invention, such as vector-based transfection, including lipid transfection or viral transfection (also known as transduction), and chemical-based transfection. However, any method for carrying nucleic acids into cells can be used. Transiently transfected cells carry / express the transfected RNA / DNA for a short period and do not passage it. Stably transfected cells continuously express and passage the transfected DNA: the exogenous nucleic acid has been integrated into the cell's genome.

[0105] As used in this article, the “DNA repair pathway” or “DRP” refers to the cellular mechanisms that allow cells to respond to the detection of DNA damage (such as single-strand or double-strand breaks) in order to maintain the integrity and function of their genome. Depending on several parameters, such as the type and length of DNA damage or the cell cycle stage at which the damage occurred, DRP refers to, but is not limited to, excision, typical homology-directed repair (typical HDR), homology recombination (HR), alternative homology-directed repair (Alt-HDR), double-strand break repair (DSBR), single-strand annealing (SSA), synthesis-dependent strand annealing (SDSA), break-induced replication (BIR), alternative end joining (Alt-EJ), microhomology-mediated end joining (MMEJ), DNA synthesis-dependent microhomology-mediated end joining (SD-MMEJ), and non-homologous end joining (NHEJ) pathways, such as typical non-homologous end joining (C-NHEJ) repair, alternative non-homologous end joining (A-NHEJ) pathway, trans-damage DNA synthesis (TLS) repair, base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), DNA damage response (DDR), blunt end joining, single-strand break repair (SSBR), interstrand crosslinking repair (ICL), and Fanconi anemia pathway (FA). However, preferably, the DRP of the present invention is selected from the group listed above.

[0106] DNA repair pathways can be suppressed, or more precisely, promoted / enhanced. Genes, mRNAs, or corresponding proteins involved in such pathways can be modulated to suppress or promote / enhance them (see examples in Table 2).

[0107] Table 2: DNA repair pathways and related genes.

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] Examples of NHEJ inhibitors (i.e., inhibitors of PARP1, Ku70 / 80, DNA-PKcs, XRCC4 / XLF, ligase IV, ligase III, XRCCl, Artemis, and PNK) include, but are not limited to, NU7441 (Leahy et al., Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. (Leahy et al., (2004)), NU7026 (Willmore et al., 2004), olaparib, DNA ligase IV inhibitor, Scr7 (Maruyama et al., 2015)), KU-0060648 (Robert et al., 2015), and anti-EGFR antibody C225 (cetuximab) (Dittmann et al.). (al., 2005), compound 401 (2-(4-morpholino)-4H-pyrimidino[2,la]isoquinoline-4-one), vanillin, Wollman penicillin, DMNB, IC87361, LY294002, OK-1035, CO 15, NK314, PI 103 hydrochloride, to name just a few exemplary inhibitors.

[0115] MMEJ inhibitors include, but are not limited to, MRE11 inhibitors, such as Mirin and its derivatives (Shibata et al, 2014), PolQ inhibitors, and CtIP inhibitors (Sfeir and Symington, 2015).

[0116] Examples of HR inhibitors include, but are not limited to, RI-1 and BO2.

[0117] Examples of HR stimulants include, but are not limited to, RS-1 (RAD51 stimulant).

[0118] NHEJ stimulants include, but are not limited to, IP6 (inositol hexaphosphate, DNA-PK enhancer, Hanakahi 2000, Ma2002, Cheung 2008).

[0119] Downregulation of DRP reduces the activity of such DRPs in cells or cell populations. Downregulation of DRP can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the repair activity (hereinafter referred to as "activity") that was not downregulated. Downregulation can be achieved in a variety of ways, such as, but not limited to, contacting the cells or cell population with one or more inhibitors (e.g., chemical inhibitors of DRP / its components), inactivating DRP / its components, downregulating DRP / its components (e.g., by contacting the cells or cell population or expressing one or more inhibitory nucleic acids, such as miRNA, siRNA, shRNA, or any combination thereof), and / or mutating one or more genes of the DRP / its components.

[0120] In a preferred embodiment, the DRP is reduced to either non-productive or competing with another DRP, hence the term competitive or non-productive approach.

[0121] For example, the NHEJ pathway can be inhibited through mechanisms such as MMEJ and related mechanisms to promote efficient integration of exogenous DNA. In the context of this invention, any active DRP can compete with another active DRP in the cell, thus constituting a competitive DR pathway. In the context of this invention, a non-productive DRP is a pathway that does not mediate the integration of exogenous DNA into the cellular genome or only inefficiently mediates its integration. For example, synthesis-dependent strand annealing (SDSA), break-induced replication (BIR), base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), DNA damage response (DDR), blunt-end joining, single-strand break repair (SSBR), and interstrand crosslink repair (ICL) are generally inefficient in mediating exogenous DNA integration.

[0122] Downregulation of a DRP typically leads to one or more other DNA repair pathways taking over the repair function of the downregulated DRP. The one or more DRPs that take over the repair function are usually upregulated. Upregulation of one or more DRPs can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of their activity without downregulation. A DRP upregulated due to the downregulation of another competing DRP is considered "promoted" (or enhanced) relative to the downregulated DRP. The degree of promotion / enhancement can be proportional to the degree of downregulation and can be, for example, an increase in activity of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% relative to the activity of the downregulated DRP without downregulation. The activity of the downregulated DRP can be diverted to one pathway, but it can also be diverted to two or more pathways that take over the DNA repair function of the downregulated DRP.

[0123] In addition to downregulating another DRP, DRP can also be upregulated by, for example, expressing one or more components of the DRP in the cells or cell population (including causing overexpression of one or more components of the DRP), introducing a component of the DRP heterologously into the cells or cell population, or by contacting the cells or cell population with one or more regulators (preferably stimulants such as chemical stimulants of one or more components of the DRP), or by mutating one or more genes of the DRP, wherein the mutation enhances the expression or activity of one or more components of the DRP.

[0124] Regulation, particularly upregulation, can typically be achieved by transiently transfecting, for example, co-transfecting (i.e., simultaneously or within one hour) cells / cell populations with one or more vectors carrying one or more genes listed in Table 2 and / or genes listed under SEQ ID NO:25-SEQ ID NO:28 and SEQ ID NO:38-SEQ ID NO:59, along with sequences having sequence identity (e.g., 99%, 98%, 97%, 96%, or 95%) with those sequences described elsewhere herein. Figure 11B The integrated vector shown is used for transfection or in use Figure 11B The integration vector shown is used in some embodiments with cell transfection time less than 24, 18, 12, 8, 4, 2, 1 hour prior to cell transfection. Figure 11B The integrative vector shown was used to transfect cells at intervals of less than 24, 18, 12, 8, 4, 2, and 1 hour. Figure 11A Some representative vectors are shown in the table. However, as those skilled in the art will understand, any gene encoding a protein involved in DRP can be used in such vectors. Some of these genes are listed in Table 2, and certain preferred genes are listed under SEQ ID NO:25-SEQ ID NO:28 and SEQ ID NO:38-SEQ ID NO:59. Figure 11AAs can be seen, certain preferred vectors insert the DRP gene between ITRs (inverted terminal repeat sequences), meaning the "sides" of the DRP gene are two ITRs, and in some preferred embodiments, genetic elements such as MARs (e.g., SGE) may also be included. Transfection is preferably performed using one or more vectors carrying MRE11, POLQ, CIRBP, and / or RAD51, for example, using one or more vectors carrying SEQ ID NO: 25 (hMRE11), SEQ ID NO: 26 (cgPOLQ), SEQ ID NO: 27 (hCIRBP), and / or SEQ ID NO: 28 (hRAD51). Other preferred DRP genes are those whose expression products are used by: (i) MMEJ and HR (Mre11, Rad50, Nbs1, CtIP, Exo1, BLM, ATM, ERCC1, Srs2, Xpf, Polδ, ligase I, ligand III), (ii) MMEJ but not HR (e.g., LigIV, XRCC1, PARP1, POLQ, WRN, POLB, Pol4) and (iii) HR but not jot MMEJ proteins (e.g., BRCA1, 53BP1, MDC1).

[0125] As used herein, a chemostimulant is a compound that can be used to enhance gene expression or protein activity. As will be readily recognized by those skilled in the art, the chemostimulant will depend on which component of the DPR (DNA repair pathway) is being stimulated. For example, RS-1 is a RAD51 stimulator that can stimulate HR. IP6 (inositol hexaphosphate) and other DNA-PK enhancers are NHEJ stimulators (see, for example, Hanakahi 2000, Ma 2002, Cheung 2008).

[0126] As used herein, a chemical inhibitor is a compound that can be used to inhibit gene expression or protein activity. Those skilled in the art will readily recognize that the chemical inhibitor will depend on which DPR is being stimulated. Examples of chemical inhibitors in the MMEJ include, but are not limited to, MRE11 inhibitors, such as Mirin and its derivatives (Shibata et al, Molec. Cell (2014) 53:7-18), PolQ inhibitors, and CtIP inhibitors (Sfeir and Symington, "Microhomology-Mediated End Joining: A Back-up Survival Mechanism or Dedicated Pathway?" Trends Biochem Sci (2015) 40:701-714). Examples of HR inhibitors include RI-1 (RAD51 inhibitor 1) and BO2 (3-(phenylmethyl)-2-[(1E)-2-(3-pyridyl)vinyl]-4(3H)-quinazolinone). See also U.S. Patent Publications 2019 / 0194694A1 and 2015 / 0361451A1.

[0127] Chemical stimulants and inhibitors are typically exogenous, meaning they are added to the cell supernatant and absorbed by the cells. Such inhibitors can be added to cells / cell populations along with the various carriers described herein, for example, simultaneously or within one hour or within less than 24, 18, 12, 8, 4, or 2 hours.

[0128] Nucleases and / or cleavage enzymes: introduction of double-strand / single-strand breaks

[0129] Different molecules are capable of introducing double-stranded and / or single-stranded breaks into genomic nucleic acids. The nucleases or cutting enzymes of this invention include, but are not limited to, homing endonucleases, restriction enzymes, zinc finger nucleases or zinc finger cutting enzymes, broad-spectrum nucleases or broad-spectrum cutting enzymes, transcription activator-like effector (TALE) nucleases or TALE cutting enzymes, guided, particularly nucleic acid-guided, nucleases or cutting enzymes such as RNA-guided nucleases or RNA-guided cutting enzymes, DNA-guided nucleases such as Argonaute (NgAgo) of Natronobacterium gregoryi or DNA-guided cutting enzymes, megaTAL nucleases, BurrH-nucleases, ARCUS nucleases, their modified or chimeric forms or variants, and combinations thereof. RNA-guided nucleases or RNA-guided cutting enzymes are optionally part of a CRISPR-based system.

[0130] In a preferred embodiment, these double-stranded and / or single-stranded breaks are introduced by one or more nucleases or cleaving enzymes. Nucleases can introduce double-stranded and / or single-stranded breaks. The term cleaving enzyme is reserved for the molecule that introduces the single-stranded break and can be a nuclease having a partially inactivated DNA cleaving domain. For example, the nuclease domains of nucleases can be mutated independently of each other to produce DNA “cleaving enzymes” capable of introducing single-stranded cuts with the same specificity as the corresponding nuclease. Due to the limitations mentioned herein, the following discussion of nucleases also applies to cleaving enzymes.

[0131] Nucleases are capable of cleaving phosphodiester bonds between nucleic acid monomers. Many nucleases participate in DNA repair by recognizing damage sites and cleaving them from the surrounding DNA. These enzymes can be part of a complex. Exonucleases are nucleases that digest nucleic acids from the ends. Preferred endonucleases herein are nucleases that act on the central region of the target molecule. Deoxyribonucleases act on DNA, and ribonucleases act on RNA. Many nucleases involved in DNA repair are not sequence-specific. However, in this context, sequence-specific nucleases are preferred. In a preferred embodiment, the sequence-specific nuclease is specific to a fairly large nucleotide string in the target genome, such as 5 or more nucleotides, or 10, 15, 20, 25, 30, 35, 40, 45, or even 50 or more nucleotides. In some embodiments, the range of target sequences in the target genome is preferably 5-50, 10-50, 15-50, 15-40, or 15-30 nucleotides. The larger such "recognition sequence" is, the fewer the target sites in the genome, and the more specific the nuclease or cleavage enzyme is to the nick formed in the genome, thus making the nick site specific. Site-specific nucleases typically have fewer than 10, 5, 4, 3, 2, or only a single (1) target site in the genome. Nucleases engineered to alter genomic nucleic acids (including by cleaving specific genomic target sequences) are referred to herein as engineered nucleases. CRISPR-based systems are one type of engineered nuclease. However, such engineered nucleases can be based on any nuclease described herein. In a preferred embodiment, the codons of the individual nucleases are optimized for expression in eukaryotic cells, such as mammalian cells. The nucleases / systems of the present invention may also include one or more linkers and / or additional functional domains, such as the terminal processing enzyme domain of a terminal processing enzyme exhibiting a 5-3' exonuclease or 3-5' exonuclease or other non-nuclease domains, such as a helicase domain.

[0132] Restriction enzymes are sequence-specific nucleases, typically specific to fairly small nucleotide strings, and therefore have short recognition sequences. The first letter of their names comes from the genus, and the last two letters from the species of the prokaryotic cell from which they were isolated. For example, EcoRI comes from the bacteria *Escherichia coli* RY13. Many restriction enzymes are restriction endonucleases, introducing, for example, straight or staggered cuts in the middle of nucleic acids. Many restriction enzymes are sensitive to the methylation state of the DNA they target. When specific bases in the enzyme recognition site are modified, the cleavage may be blocked or impaired.

[0133] Examples of important methylation-sensitive restriction enzymes in epigenetics include DpnI and DpnII, which are sensitive to the detection of N6-methyladenine at the GATC recognition site, and HpaII and MspI, which are sensitive to the detection of C5-methylcytosine at the CCGG recognition site.

[0134] Some of the exemplary restriction enzymes used in the examples are listed in Table 3 along with their recognition sites, their CpG methylation sensitivities, and the number of target sites found in the reference CHO genome.

[0135] Table 3: Examples of restriction enzymes and their target sites in the CHO genome.

[0136]

[0137] Nucleases that recognize sequences longer than 12 base pairs are called macronucleases. Macronucleases / -cleaving enzymes are deoxyribonucleases characterized by a large recognition site (e.g., a double-stranded DNA sequence of 12 to 40 base pairs, such as 20 to 40 or 30 to 40 base pairs); therefore, this site may appear only once in any given genome.

[0138] "Homing endonucleases" are a type of broad-spectrum nuclease, a double-stranded DNAase with a large asymmetric recognition site and a coding sequence that is usually embedded in an intron or protein intein. Homing endonucleases recognize extremely rare sites in the genome, thus they cleave at very few locations, sometimes even at a single location (WO2004067736, see also US8697395 B2).

[0139] Zinc finger nucleases / -cleavage enzymes (ZFNs) are artificial restriction enzymes created by fusing a zinc finger DNA-binding domain with a DNA-cleaving domain. The zinc finger domain can be engineered to target specific desired DNA sequences. For example, ZFN described by Urnov F ​​et al. (Highly efficient endogenous human gene correction using designed zinc-finger nucleases (2005) Nature 435:646-651).

[0140] Transcription activator-like effector (TALE) nucleases / cleavage enzymes are restriction enzymes that can be engineered to cleave specific DNA sequences. Transcription activator-like effectors (TALEs) can be engineered to bind to virtually any desired DNA sequence, so that when bound to a DNA cleavage domain, the DNA can be cleaved at a specific location. Examples include TALE nucleases described by Mussolino et al. (A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity (2011) Nucl. Acids Res. 39(21):9283-9293).

[0141] RNA-guided nucleases / -cutting enzymes, particularly endonucleases, include, for example, Cas9 or Cpf1. CRISPR systems have been described in detail. Any CRISPR-based system is part of this invention. In the case of using another RNA-guided endonuclease, a suitable guide RNA, sgRNA, or crRNA, or other suitable RNA sequence, that interacts with the RNA-guided endonuclease and targets genomic target sites in genomic nucleic acids, can be used.

[0142] In some preferred embodiments, the nuclease is an RNA-guided nuclease. Non-limiting examples of RNA-guided nucleases (including nucleic acid-guided nucleases) used in this disclosure include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csxl2), Cas10, CasX, CasY, Cpf1, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, C sm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cms1, Cpf1, their homologs, their orthologues, or their modified forms, MAD7 such as MADzyme (INSCRIPTA), C2cl, C2c2, C2c3.

[0143] In some preferred embodiments, the nuclease is a DNA-guided nuclease. "DNA-guided nuclease" refers to a system comprising a DNA guide (gDNA) and a nuclease. The DNA guide, such as 5'-phosphorylated single-stranded DNA (ssDNA), guides the nuclease to cleave double-stranded DNA targets within the DNA-guided cleavage enzyme. "Argonaute-based system" refers to a DNA-guided nuclease based on a single-stranded DNA guide (gDNA) and a nuclease from the Argonaute (Ago) protein family. The gDNA targets the nuclease to a specific DNA sequence, resulting in sequence-specific DNA cleavage. The Ago protein can be mutagenized to exhibit higher activity at 37°C. Several Argonaute proteins were characterized as originating from Natronobacterium gregoryi Argonaute (see, for example, Gao et al., DNA-guided genome editing using the Natronobacterium gregoryi Argonaute, Nature Biotechnology, published online May 2, 2016), Rhodobacter sphaeroides (RsAgo, for example, Olivnikov et al.), Thermo thermophiles (TtAgo, for example, Swarts et al. (2014), Nature 507(7491):258-261), and Pyrococcus furiosus Argonaute (PfAgo).

[0144] Argonaute-based systems can be used to target and cut genomic DNA within cells.

[0145] “TtAgo” is a prokaryotic Argonaute protein that is thought to be associated with gene silencing. TtAgo is derived from thermophilic bacteria. (See, for example, Swarts et al, ibid, G. Sheng et al, (2013) Proc. Natl. Acad. Sci. USA III, 652).

[0146] One of the most well-known prokaryotic Ago proteins comes from thermophilic bacteria (TtAgo; Swarts et al., ibid.). This TtAgo-bound "guide DNA" directs the protein-DNA complex to bind to Watson-Crick complementary DNA sequences in third-party DNA molecules. Once the sequence information in these guide DNAs allows recognition of the target DNA, the TtAgo-guide DNA complex cleaves that target DNA. This mechanism is also supported by the structure of the TtAgo-guide DNA complex when it binds to its target DNA (G. Sheng et al., ibid.). Ago from Rhodophyta spp. (RsAgo) exhibits similar properties (ibid.).

[0147] Exogenous guide DNA of any DNA sequence can be loaded onto the TtAgo protein (Swarts et al., ibid.). Since the specificity of TtAgo cleavage is determined by the guide DNA, the TtAgo-DNA complex formed with the exogenous, researcher-specified guide DNA will guide the TtAgo target DNA to cleave into complementary researcher-specified target DNA. In this way, targeted double-strand breaks can be generated in DNA. The use of a TtAgo-guide DNA system (or an orthologous Ago-guide DNA system from another organism) allows for targeted cleavage of genomic DNA within cells. This cleavage can be single-stranded or double-stranded. For cleavage of mammalian genomic DNA, it is preferable to use an optimized version of the TtAgo codon expressed in mammalian cells. Furthermore, it is preferable to treat cells with a TtAgo-DNA complex formed in vitro, wherein the TtAgo protein is fused to a cell-penetrating peptide. Ago-RNA-mediated DNA cleavage can be used to achieve a range of effects, including gene knockout, target gene addition, gene correction, and target gene deletion, using technical standards in the art for the development of DNA breaks.

[0148] Illustrative examples of Argonaute-based systems and gDNA designs are disclosed in WO 2017 / 107898, CN105483118, WO 2017 / 139264, U.S. Patent Applications Nos. 2017367280 and 20180201921, and their cited references, all of which are incorporated herein by reference in their entirety. Argonaute-based systems optionally include one or more adapters and / or additional functional domains, such as a terminal processing enzyme domain of a terminal processing enzyme exhibiting a 5–3' exonuclease or 3–5' exonuclease, or other non-nuclease domains, such as a helicase domain.

[0149] “megaTAL nuclease / megaTAL cleaving enzyme” refers to an engineered nuclease containing an engineered TALE DNA-binding domain and an engineered macronuclease or engineered homing endonuclease. The TALE DNA-binding domain can be designed to bind DNA at virtually any locus in a nucleic acid sequence in the genome, and if this DNA-binding domain is fused with an engineered macronuclease, it can cleave the target sequence. Illustrative examples of megaTAL nucleases and the design of TALE DNA-binding domains are disclosed, for example, in the description by Boissel et al. (MegaTALs: a rare-cleaving nuclea researchitecture for therapeutic genome engineering (2013), Nucleic Acids Research 42(4):2591-2601) and the references cited therein, all of which are incorporated herein by reference in their entirety. megaTAL nucleases optionally include one or more linkers and / or additional functional domains, such as C-terminal domain (CTD) peptides, N-terminal domain (NTD) peptides, terminal processing enzyme domains of terminal processing enzymes that exhibit 5-3' exonuclease or 3-5' exonuclease, or other non-nuclease domains, such as helicase domains.

[0150] The “TALE DNA-binding domain” is the DNA-binding portion of transcription activator-like effectors (TALE or TAL effectors) that mimics plant transcription activators to manipulate the plant transcriptome (see, for example, Kay et al., 2007. Science 318:648-651). In specific implementation schemes, the TALE DNA-binding domains anticipated are either engineered from scratch or derived from naturally occurring TALEs, including but not limited to AvrBs3 from Xanthomonas campestris pv. vesicatoria, Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfa, Xanthomonas citri, Xanthomonas euvesicatoria, and Xanthomonas oryzae, and brgl 1 and hpxl7 from Ralstonia solanacearum. Illustrative examples of TALE proteins for deriving and designing DNA-binding domains are disclosed in U.S. Patent No. 9,017,967 and the references cited therein, all of which are incorporated herein by reference in their entirety.

[0151] "BurrH-nuclease" refers to a fusion protein with nuclease activity containing a modular base-per-base specific nucleic acid binding domain (MBBBD). These domains are derived from proteins of the bacterial endosymbiont Burkholderia Rhizoxinica or other similar proteins identified in marine organisms. By combining different modules of these binding domains, the modular base-per-base binding domain can be engineered to have binding properties to specific nucleic acid sequences, such as DNA-binding domains. Such engineered MBBBDs can then be fused with nuclease catalytic domains to cleave DNA at virtually any locus in the nucleic acid sequence of the genome. Illustrative examples of BurrH-nucleases and the design of MBBBDs are disclosed in WO 2014 / 018601 and US2015225465 A1, and the references cited therein, all of which are incorporated herein by reference in their entirety. BurrH-nucleases optionally include one or more linkers and / or additional functional domains, such as the terminal processing enzyme domain of a terminal processing enzyme that exhibits a 5-3' exonuclease or a 3-5' exonuclease, or other non-nuclease domains, such as a helicase domain.

[0152] In the context of the disclosed methods and cells, enzymes such as transposases or integrases can also be used as cleavage enzymes / nucleases.

[0153] Targeting elements for targeting at least one locus in a cell genome containing an insertion site of an endogenous retroviral (ERV) sequence or an LTR-retrotransposon (LTR-RT) sequence are typically sequences that promote and / or guide the activity of cleavage enzymes and / or nucleases. Such targeting elements include, for example, guide RNAs, including single guide RNAs (sgRNAs) or crRNAs (CRISPR RNAs), and are encoded by CRISPR and Cas9, Cpf1, or Cms1 nuclease expression vectors, for example, targeting the ERV C109F 5' genome sequence (SEQ ID 8 and SEQ ID 10) and the ERV C109F 3' genome sequence (SEQ ID 9 and SEQ ID 11). The DRP that can be upregulated and / or downregulated can be adjusted depending on the type of element used. For example, for DRPs upregulated by CRISPR cleavage sites 16 and 17 (see CRISPR cleavage site 17), the DRP can be adjusted accordingly. Figure 5A and Figure 5BThe DSB generated by the gene is upregulated via homologous recombination (HR) in some embodiments. Furthermore, the vector carrying the transgene may include 5' and 3' homologous arms (SEQ ID 6 and SEQ ID 7), which are present in the vector and locus including the insertion site.

[0154] The sequence specificity of the CRISPR (clustered regularly spaced short palindromic repeats) system is determined by small RNAs. CRISPR loci consist of a series of repeat sequences separated by "spacer" sequences, which match the genomes of bacteriophages and other mobile genetic elements. The repeat spacer array is transcribed into a long precursor and processed within the repeat sequences to generate small crRNAs that specify the target sequence (also known as the prespacer sequence) to be cleaved by the CRISPR system. For cleavage, a sequence motif, called a protospacer-adjacent motif (PAM), is typically required directly downstream of the target region. CRISPR-associated (cas) genes are usually located on the flanks of the repeat spacer array and encode the enzymatic mechanisms responsible for crRNA (CRISPR RNA) biogenesis and targeting. For example, Cas9 is a dsDNA endonuclease that uses crRNA guides to specify the cleavage site. Loading the crRNA guide onto Cas9 occurs during the processing of the crRNA precursor and requires small RNAs, tracrRNAs, and RNAse III, which are antisense to the precursor. Compared to genome editing using ZFN or TALEN, altering the target specificity of Cas9 does not require protein engineering; it only requires designing short crRNA guides, which are also known as sgRNAs when fused with tracrRNA (trans-activating CRISPRRNA).

[0155] To date, three different types of Cas9 nucleases (e.g., Cas9) have been used in genome editing protocols. The first is wild-type Cas9, which can perform site-specific cleavage of double-stranded DNA, thereby activating the double-strand break (DSB) repair mechanism. DSB can be repaired via the non-homologous end joining (NHEJ) pathway, resulting in insertions and / or deletions (indels) that disrupt the target locus. Alternatively, if a donor template homologous to the target locus is provided, DSB can be repaired via the homology-directed repair (HDR) pathway, allowing for precise substitution mutations.

[0156] The Cas9 system has been further engineered to improve precision by developing a mutant form called nCas9, possessing only cleavage enzyme activity (e.g., Cas9D10A). This means it cuts only one DNA strand and does not activate the NHEJ. Instead, when a homology repair template is provided, DNA repair proceeds only via the high-fidelity (HDR) pathway, reducing insertion and / or deletion mutations. Therefore, Cas9D10A is more attractive in terms of target specificity in many applications when the locus is designed to generate paired Cas9 complexes targeting adjacent DNA cleavages. Such Cas cleavage enzymes can also be fused with other functional or catalytic domains, such as domains that provide deamination activity (e.g., for base editing purposes).

[0157] The third type is based on enzyme inactivation of Cas9 (eiCas9), also known as inactivated Cas9 endonuclease (inactivated Cas9 or dCas9). This system contains a Cas9 mutant that lacks endonuclease activity due to mutations in its endonuclease domains (e.g., RuvC and HNH domains). dCas9 can still bind to its guide RNA and DNA strand and can be fused with functional or catalytic domains, providing domains selected from, but not limited to, DNA modification activities such as: nuclease activity (e.g., Fok1), Clo51, methyltransferase activity, demethylase activity, deamination activity, depurination activity, integrase activity, transposase activity, and recombinase activity. Other domains that provide protein modification activities include, but are not limited to, repression domains (e.g., KRAB domains), activation domains (e.g., VP16), methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristylation activity, demyristylation activity, glycosylation activity, and deglycosylation activity.

[0158] The term sequence identity refers to a measure of the consistency of a nucleotide or amino acid sequence. Typically, sequences are aligned to obtain the highest possible order match. "Identity" itself has a recognized meaning in the art and can be calculated using publicly available techniques. (See, e.g.: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991). Although there are many methods to measure the identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to technicians because it defines the same nucleotide or amino acid at a given position in the sequence (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)).

[0159] Initial sequence alignment can be performed using known computer programs such as DNAsis software (Hitachi Software, SanBruno, CA), followed by multiple sequence alignment using ESEE version 3.0 DNA / protein sequence software to determine whether any particular nucleic acid molecule is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the gamma retrovirus-like sequences or portions thereof of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0160] Known computer programs such as BESTFIT (Wisconsin Sequence Analysis) can be used conventionally. Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) determines whether the amino acid sequence is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a protein expressed, for example, by SEQ ID NO:1 or SEQ ID NO:3 or a portion thereof. BESTFIT uses the local homology algorithm from Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the optimal homologous fragment between two sequences.

[0161] When using DNAsis, ESEE, BESTFIT, or any other sequence alignment program to determine whether a particular sequence has, for example, 95% similarity to a reference sequence according to the present invention, parameters are set to calculate the percentage of similarity in the full length of the reference nucleic acid or amino acid sequence, and homology gaps are allowed up to 5% of the total number of nucleotides in the reference sequence.

[0162] Another preferred method for determining the best overall match between the query sequence (the sequence of this invention) and the target sequence, also known as global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In sequence alignment, both the query sequence and the target sequence are DNA sequences. RNA sequences can be compared by converting U to T. The result of the global sequence alignment is a percentage of consistency. Preferred parameters for calculating the percentage of consistency in FASTDB alignment of DNA sequences are: matrix = single, k-tuple = 4, mismatch penalty = 1, connection penalty = 30, length of randomization group = 0, abort score = 1, gap penalty = 5, gap size penalty = 0.05, window size = 500 or the length of the target nucleotide sequence, whichever is shorter.

[0163] For example, a polynucleotide having 95% “identity” with the reference nucleotide sequence of the present invention is identical to the reference sequence, except that the polynucleotide sequence may include an average of up to five point mutations per 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a polynucleotide having at least 95% identity with the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted, or up to 5% of the nucleotides in the reference sequence may be replaced with another nucleotide, or some nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be a complete sequence, an ORF (open reading frame), or any fragment specified as described herein.

[0164] The NCBI Local Sequence Alignment Retrieval Tool (BLAST) (Altschul et al. J. Mol. Biol. 215:403-410, 1990) is available from multiple sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and online, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. It is available on the NCBI website, along with instructions on how to use the program to determine sequence identity and sequence similarity.

[0165] This invention refers not only directly to sequences that have a specific sequence identity with the sequences disclosed herein, but also to sequence variants of any sequences disclosed herein. Therefore, this invention also refers to sequence variants in any context that refers to a specific sequence identity, and vice versa. A “sequence variant” is a polynucleotide or polypeptide that differs from the sequence (polynucleotide or polypeptide sequence) disclosed herein but retains its essential characteristics. Typically, variants are very similar to the sequences disclosed herein and are identical in many regions.

[0166] The variants may contain changes to coding regions, non-coding regions, or both. Particularly preferred are sequence variants containing substitutions, additions, or deletions that produce a silencing effect but do not alter the properties or activity of, for example, the encoded polypeptide. Due to the degeneracy of the genetic code, nucleotide variants resulting from silencing substitutions are preferred. Furthermore, variants with any combination of substitutions, deletions, or additions of 5 to 10, 1 to 5, or 1 to 2 amino acids are also preferred.

[0167] The amino acid sequence of the variant peptide may differ from that described in SEQ ID NO: 3 by the insertion or deletion of one or more amino acid residues and / or the substitution of one or more amino acid residues with different amino acid residues. Preferably, the amino acid changes are minor in nature, i.e., conserved amino acid substitutions that do not significantly affect, for example, protein folding and / or activity; small deletions (typically 1 to about 30 amino acids); small extensions to the N-terminus or C-terminus, such as N-terminal methionine residues; small linker peptides of up to about 20 to 25 residues; or small extensions that facilitate purification by altering net charge or other functions such as multihistidine bundles, antigenic epitopes, or binding domains. Examples of conserved substitutions include those within the group of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that do not typically alter specific activity are known in the art and described, for example, in H. Neurath and RL Hill, 1979, in *The Proteins*, Academic Press, New York. The most common exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, and the reverse.

[0168] A "sequential nucleotide" at a certain percentile refers to nucleotides that follow each other directly. Therefore, 10% of the nucleotides in SEQ ID NO:2, which contains 60,000 nucleotides, could be nucleotides 1-6000 or nucleotides 2-6001, etc.

[0169] Gene silencing via, for example, siRNA has been described elsewhere, such as in U.S. Patent Publication 20180016583, which is incorporated herein by reference in its entirety, particularly its disclosure and gene silencing.

[0170] Example

[0171] The purpose of the following examples is firstly to identify the transgene insertion locus, here through CRISPR-mediated cleavage (Figure 2), and secondly to compare the effect of homology on transgene sequences with integrated loci with the effect of non-homology on transgenes. Figure 3A , Figure 3B , Figure 4A , Figure 4B , Figure 5A , Figure 5B Furthermore, DNA repair pathways such as homologous recombination (HR) or alternative end joining (Alt-EJ) were compared. Figure 6 The effects of upregulation / downregulation and stimulation / inhibition of )

[0172] Table 4: Experiment Summary.

[0173]

[0174] Set 1: No DNA homology / Alt-EJ stimulation / NHEJ inhibition

[0175] Set 2: No DNA homology / No Alt-EJ stimulation / NHEJ inhibition

[0176] Set 3: DNA homology / HR stimulation / NHEJ inhibition

[0177] Set 4: DNA homology / no HR stimulation / NHEJ inhibition

[0178] Example 1: Targeted integration into the ERV C109F locus

[0179] This example illustrates the integration of the transgene into the ERV C109F locus (SEQ ID NO:1, SEQ ID NO:2).

[0180] CHO-M cells were transfected with a vector targeting the ERV C109F locus in each genome. Figure 3A , Figure 3B , Figure 4A , Figure 4B Such vectors either do not contain homologous sequences (). Figure 3A , Figure 3B ), or contain homologous sequences ( Figure 4A , Figure 4B For both the "non-homologous" and "homologous" approaches, the chemical inhibitor Nu7441 was used to inhibit the non-homologous end-joining (NHEJ) DNA repair pathway. Furthermore, each approach included in the experiments was designed to assess the presence or absence of homologous recombination (HR) or alternative end-joining (Alt-EJ) stimulation.

[0181] For both methods, there are four possibilities for transgene integration in the identified ERV C 109F locus: i) no transgene integration, or ii) integration at the WT allele of the ERV C 109F locus, or iii) integration at the ERV-C 109F allele, or finally iv) integration at both alleles (ii) and (iii), which is sometimes referred to as “two loci” in this paper.

[0182] These results were obtained using three TaqMan qPCR analyses to determine the class of each clone. Figure 5A and Figure 5B These analyses are explained in the context of Figure 12.

[0183] Figure 6 The percentage of clones belonging to categories (i), (ii), (iii), and (iv) as defined above is shown. This figure specifically illustrates the targeting efficiency of CRISPR targeting compared to random integration. Clones obtained through non-targeted integration correspond to clones exhibiting fluorescent signals similar to those obtained from CHO-M wild-type cells: this infers that transgene integration occurred in a random genomic sequence, as it did not target the ERV locus.

[0184] In this embodiment, it is noted that after HR stimulation and NHEJ inhibition, integration of DNA-homogeneous transgenes resulted in a higher integration frequency in the ERV-containing alleles than in the non-DNA-homogeneous alleles. This may reflect the fact that homology facilitates targeted transgene integration through homologous recombination. However, when using an expression vector without homology but with Alt-EJ stimulation and NHEJ inhibition, the targeted integration frequency of both alleles was highest. Figure 6 ).

[0185] In summary, this paper presents an efficient method for transgene integration using CRISPR and gRNA: 70% to 90% higher targeted integration rates were observed compared to non-targeted integration. Furthermore, in this embodiment, stimulation via the HR or Alt-EJ pathway did indeed increase the efficiency of targeted integration into ERV-containing alleles or both alleles, depending on the presence or absence of DNA homology.

[0186] Figure 7A and Figure 7B Histograms of the chromosomal distribution frequencies of DNA-FISH signals in sets 1 and 3 are shown (set composition is shown in Table 4): Cells from sets 1 and 3 were blocked in metaphase using colchicine and seeded on slides. DNA-FISH experiments were then performed on each sample using probes targeting promoters that drive GFP (green fluorescent protein) expression; images were acquired using a confocal microscope (Zeiss LSM800). Finally, the images were analyzed and karyotypes were generated using a karyotype analyzer. Targeted integration in chromosomes 15 (Chr15) and 9 (Chr9) was enriched up to 60% and 19.5% in set 1, and up to 45% and 32% in set 3, respectively. The n-value represents the number of karyotypes analyzed.

[0187] In both sets, several transgene integration sites were observed on other chromosomes, but at a much lower frequency, since total random transgene integration events account for about 20%.

[0188] Finally, the results confirm that CRISPR cleavage combined with NHEJ inhibition and HR or, in particular, Alt-EJ MMEJ mechanism activation can achieve efficient targeted transgene integration on chromosomes 15 and 9.

[0189] These results indicate that targeted integration can occur efficiently upon Alt-EJ activation, with up to 80% of integration occurring on portions of chromosome 15 and / or chromosome 9 containing two alleles of the ERV-109F locus (i.e., the ERV-deleted WT and the allele containing ERV C 109F). CHO-M cells lacked homologous sequences at all chromosomal loci on their homologous chromosomes because, like all CHO cells, they underwent extensive chromosomal rearrangements during selection for optimal in vitro growth characteristics after isolation from native Chinese hamster cells. This explains why homologous sequences in the genome can be located on different chromosomes, as observed at the loci examined.

[0190] Figure 7C Representative karyotypes of transgene insertions occurring in chromosomes 9 and 15 from cells in set 1 are shown, with DNA stained gray and DNA-FISH probes targeting telomeric repeat sequences (sequence TTAGGGTTAGGGTTAGGG [SEQ ID NO:60]) and the promoters driving GFP expression stained white. White arrows indicate the locations of FISH signals for the ERV and WT alleles.

[0191] Example 2: Increasing transgene expression by targeting the ERV C109F locus

[0192] This example illustrates that the ERV C109F locus allows for enhanced and stable expression of exogenous transgenes. Figure 8 shows the analysis of GFP-expressing CHO clones based on FITC fluorescence analysis. 340,000 cells were transfected under each condition and plated on semi-solid medium with antibiotic selection two days post-transfection. 42 clones were picked based on fluorescence intensity. They were then cultured. Nine days after selection, the FITC levels of 2000 cells from each clone were measured. And analyzed by qPCR The type of transgene integration event for each clone was determined. The 42 clones were divided into four categories: transgenes located either in wild-type alleles, in alleles containing ERV C109F, in both alleles, or in non-targeted integration corresponding to off-target genome integration events, as previously described.

[0193] Figure 8A , Figure 8B , Figure 8C and Figure 8D Fluorescence intensity (FITC) depending on the type of transgenic locus integration is displayed. Fluorescence representation for each integration type allows for examination of whether a locus provides higher transgenic expression compared to other loci, and whether regulation of repair pathways alters transgenic expression levels.

[0194] Global results indicate that targeting the ERV C109F locus appears to mediate higher FITC fluorescence compared to random genomic integration, which is represented by non-targeted integration. Furthermore, it can be observed that integration of the wild-type allele at the ERV C109F locus produces higher fluorescence levels than the ERV allele during DNA repair pathway regulation (e.g., FITC fluorescence from clones isolated from sets 1 and 3). Figure 8A and Figure 8C Integration into the wild-type allele at the ERV C109F locus also produced higher fluorescence compared to integration into both alleles. Therefore, in certain embodiments of the invention, integration into the wild-type allele at the ERV C109F locus is preferred over integration into the corresponding ERV-occupied allele. This embodiment is particularly preferred if potential negative effects might occur after integration into the ERV-containing allele, and / or if the WT ERV-deleted allele is more conducive to high and stable transgene expression than the ERV-containing allele. As those skilled in the art will understand, also in embodiments where no transgene integration occurs in the ERV-containing allele, the ERV sequence is modified in some embodiments to eliminate or reduce the release of viral particles from the cell, preferably such that the release of viral particles is no longer detectable (see Duroy, 2020).

[0195] In some implementations, regulation of the DNA repair pathway is also particularly preferred because it also appears to provide enhanced transgene expression at this locus, especially when Alt-EJ regulation is applied, which produces significantly higher expression than without such regulation (see, for example, in...). Figure 8A and Figure 8B (The dashed line in the middle represents the median fluorescence value).

[0196] Figure 9The results are shown from another transfection and another batch of clones obtained from sets 3 and 4. These results demonstrate that when comparing cells generated using HR regulation (set 3, set 4 ... Figure 8C ) and cells produced without HR regulation (set 4, Figure 8D Fluorescence obtained from clones generated using targeted integration with DNA homology.

[0197] These results validate previous findings, demonstrating that regulation of the HR repair pathway can also be used to generate cell clones exhibiting increased transgene expression, particularly when the transgene integrates into the WT allele or into both alleles, compared to integration only into the ERV-containing allele or non-targeted integration. This further validates that regulation of DNA repair mechanisms favors increased transgene expression.

[0198] Example 3: Expression of complex proteins

[0199] This example demonstrates that a setting that allows GFP expression when integrated into the ERV109F-deleted WT allele on chromosome 9 and / or the ERV109F-containing allele on homologous chromosome 15 (Figure 10) also allows the expression of trastuzumab therapeutic protein.

[0200] Initially, expression vectors for CRISPR-mediated cleavage were used, along with trastuzumab expression vectors, to transfect CHO cells. Figure 11B For some transfections, vectors mediating the expression of POLQ, MRE11, and CIRBP proteins are also included, which enhance alternative terminal joining mechanisms, such as the microhomology-mediated terminal joining (MMEJ) pathway. Figure 11B The aim of this study was to facilitate the integration of trastuzumab expression vectors into the CRISPR cleavage sites on the chromosome, as trastuzumab expression vectors do not possess homologous sequences and therefore do not exhibit significant DNA sequence homology with chromosomal loci. Cells containing transgenic sequences with genomic integration were selected for puromycin antibiotic resistance, and clonal populations were then isolated using a ClonePix FL imager from Molecular Devices, LLC.

[0201] Then, using the primers indicated by the arrows in Figure 12, the derived clones were analyzed by PCR to determine which genomic locus the transgene was integrated into: Figure 12B The amplification performed using the primers indicated by the middle arrow shows that no transgene insertion occurred in the ERV-deleted WT allele (referred to as the ERV-free locus in the figure). Figure 12C The primers shown assessed whether the ERV was still inserted at its regular locus without transgene insertion. (Using...) Figure 12DThe amplification performed using all the primers shown indicates that both alleles are intact, and that transgene integration must have occurred elsewhere in the genome. Figure 12A In the study, the absence of amplification by either primer pair indicated the presence of insertion in both alleles. This led to the identification of four types of clones, including transgenes integrated at random locations, in the ERV109F allele on chromosome 15, in the ERV109F-deleted WT allele on chromosome 9, or integrated at both alleles on chromosomes 15 and 9. As expected, clones with the Tras transgene integrated at the ERV-containing allele or both alleles, thus replacing the ERV109F sequence with the Tras coding sequence, showed the largest reduction in ERV expression, up to 17-fold or more, reaching undetectable levels in the PCR background. Figures 13A-13C ).

[0202] Then, clones were screened based on trastuzumab secretion, and for each clone type, those with low levels ( Figure 14A ), moderate ( Figure 14B ) and high level ( Figure 14C Representative clones expressing trastuzumab. As observed for GFP expression, the highest levels of trastuzumab production were obtained when the Tras coding sequence was integrated into the ERV-deleted chromosome 9 locus, followed by clones with Tras sequences integrated into both loci. Production levels obtained from targeted integration containing the ERV109F allele and / or the ERV109F-deleted WT allele were significantly higher than those obtained from random genomic integration. Overall, these findings suggest that the genomic environment at the ERV locus is highly favorable for gene expression, and that the highest-yielding clones were obtained when the transgene was integrated into the ERV-deleted chromosome 9 WT allele.

[0203] Next, the high Tras titers obtained in the supernatants of small-scale and short-term non-feedable cultures were assessed for their potential to be converted into therapeutic protein production-like cultures. Specific productivity levels obtained over 6 to 10-day intervals in small-scale 96-well plates were evaluated. Figures 15D-15F ), and the titer obtained from the supernatant of fed-batch cultures in a shaken 24-well plate ( Figures 15A-15C This indicates that optimal Tras expression was obtained by integrating the transgene into the ERV-deleted WT allele.

[0204] Expanding scale

[0205] Used in Fostered culture in 15 bioreactors was used to evaluate the productivity of clones mediating the highest titers of each genome integration on a larger scale. Figure 15Cand Figure 15F For clones that integrated the transgene at the ERV-deleted wt allele, a specific productivity of over 20 picograms of secreted Tras IgG per cell per day was achieved. Figure 16B This clone also mediates the release of the highest titer of antibody into the cell culture supernatant. Figure 16A ).

[0206] Overall, a surprising finding is that the optimal targeted integration locus for high transgenic expression and the production of therapeutic proteins is a highly expressed ERV-integrated chromosomal locus, more preferably the chromosome 15 genomic allele containing ERV109F, and even more preferably the ERV109F-deleted WT allele on chromosome 9. Expression vectors for alternative terminal linker factors such as MRE11 and PolQ MMEJ proteins (see...) Figure 11A Co-transfection with therapeutic protein vectors can facilitate higher-frequency targeted integration of non-homologous plasmid sequences at this favorable genomic locus. Dual integration of the therapeutic protein expression vector at both the ERV allele and the ERV-deleted WT allele is also highly advantageous, allowing for high-level production of the therapeutic protein with a single transfection and eliminating the expression of potentially harmful retroviral sequences that would lead to the release of viral particles along with the therapeutic protein of interest into the cell supernatant.

[0207] Materials and Methods

[0208] Cell culture

[0209] Suspension-adapted Chinese hamster ovary (CHO-M) derived cells were maintained in serum-free BalanCD CHO medium (Irvine Scientific) supplemented with L-glutamine (GE Healthcare). CHO-M viable cell density and fluorescence signal of green fluorescent transfected cells were assessed using a Cytoflex flow cytometer (Beckman Coulter). Cells were cultured in 50 ml C50 bioreactor tubes (TPP, Switzerland) in a humidified incubator at 37°C and 5% CO2 at 180 rpm, and passaged every 3–4 days.

[0210] plasmid construction

[0211] This study used two EGFP (enhanced green fluorescent protein) expression vectors. Both vectors shared the same eukaryotic expression cassette, consisting of an antibiotic resistance cassette followed by an EGFP expression cassette with a downstream SV40 enhancer and a SELEXIS genetic element (SGE). SELEXIS SGEs are unique DNA-based epigenetic elements that control the dynamic organization of chromatin in all mammalian cells. They enhance transcription by dissociating integrated transgenes from the silencing effects of surrounding chromatin.

[0212] Duroy et al. (2019) described that of the 173 type C ERVs identified in the CHO genome, only one could be transcribed and produce viral particles present in the CHO culture supernatant. The integrated locus was specific because the ERV sequence existed only in a hemizygous state in the CHO cell genome. Figure 1 This means that one allele exists in the CHO genome without ERV integration (called the wild-type (WT) or ERV-deleted WT allele), while another allele exists in a homologous DNA sequence within the CHO genome where the integrated ERV can be found (called the allele containing ERV-C 109F, or the ERV-C109F allele). Only the latter allele will express the corresponding viral sequence in the cell, resulting in the presence of ERV-C 109F mRNA in the cytoplasm.

[0213] In addition, one of the vectors with EGP used in this study contains two additional 750 bp homologous sequences that correspond to two DNA sequences (SEQ ID 6 and SEQ ID 7) from genomic loci surrounding the ERV C 109F allele and the ERV-deleted WT allele. These sequences are located on each side of the allele breakpoint and the CRISPR cleavage site (5' and 3' homologous arms), as shown in Figure 2.

[0214] In addition to the EGFP vector, two sets of CRISPR vectors were used to introduce site-specific DSBs. Preferably, CRISP16 and CRISP 17 DSBs (SEQ ID 8 and SEQ ID 9) were repaired via homologous recombination using 5' and 3' homologous arms, which are present as homologous sequences in the WT alleles and / or ERV-containing alleles of the vector and locus. Preferably, CRISP 50 and CRISP 51 DSBs (SEQ ID 10 and SEQ ID 11) were repaired via the Alt-EJ pathway using microhomologous sequences present in the vector and wild-type alleles.

[0215] Transfection and single-cell isolation

[0216] To inhibit the NHEJ DNA repair pathway, suspension-grown CHO-M cells were pretreated with 0.5 μM Nu7441 to suppress DNA-PKcs. Cells stimulated by the homologous recombination (HR) pathway were also treated with 1 μM RS-1. As shown in Figures 3 and 4, pretreated cells (340,000 cells / transfection) were transfected with two expression vectors containing the encoding sequence of enhanced green fluorescent protein (EGFP) for detection. To stimulate the Alt-EJ repair pathway, i.e., the MMEJ pathway, cells were also transfected with the hMRE11 (SEQ ID 25), cgPOLQ (SEQ ID 26), and hCIRBP (SEQ ID 27) genes cloned into individual expression vectors. However, to stimulate the HR repair pathway, cells were also transfected with the hMRE11 and hRAD51 (SEQ ID 28) genes cloned into individual expression vectors.

[0217] One day after transfection, cells were centrifuged and the culture medium was replaced to remove Nu7441 and RS-1. Two days after transfection, cells were seeded at a density of 5000 cells / ml on semi-solid medium containing 3 μg / ml puromycin. After 10 days of growth in semi-solid medium, 42 clones / transfected cells expressing EGFP (ClonePix, MolecularDevices) were selected based on fluorescence intensity and cultured in BalanCD CHO medium. EGFP expression levels (FITC) were measured in 2000 cells from each clone 9 days after selection (after DNA repair pathway stimulation) and 6 days after selection (after no DNA repair pathway stimulation). Results were analyzed by qPCR. The identified categories are displayed.

[0218] qPCR analysis

[0219] DNA extraction

[0220] Following the manufacturer's instructions, use CellsDirect One-Step qRT-PCR. (ThermoFisher Genomic DNA (gDNA) was extracted from 2 × 10⁶ cells. Using... Spectrophotometer (ThermoFisher) ) Perform gDNA quantification.

[0221] qPCR analysis

[0222] Three types were designed qPCR analysis Figure 5A and Figure 5B (Probes and primers are described in SEQ ID NO:29 to SEQ ID NO:37). The linearity and efficiency of all TaqMan qPCR analyses were validated using a standard curve method. All analyses were highly linear (=0.999) and showed very good efficiency (≥0.97).

[0223] qPCR was run on QIAGEN's Rotor-Gene, using Rotor-Gene Multiplex PCR. and FAM or HEX marked qPCR analysis was performed using Rotor-Gene Q. The software (v2.3.1) is used for data analysis.

[0224] Specificity for validation of the three loci was determined using appropriate negative controls and a standard curve method. qPCR analysis was performed to verify the presence or absence of the reference locus.

[0225] The presence or absence of an amplicon within the CT range corresponding to the control allows determination of whether the target locus of the ERV-integrated allele or the target locus in the wild-type allele of the ERV-integrated locus is the same as in untransfected CHO-M cells. The "yes or no" results of three different TaqMan analyses can determine which category each clone belongs to.

[0226] Fluorescence in situ hybridization experiment

[0227] Cells were blocked at metaphase using colchicine and spread on glass slides. DNA-FISH experiments were performed on each sample using a probe targeting the promoter driving EGFP (green fluorescent protein) vector expression. Images were acquired using a Zeiss LSM800 confocal microscope. Finally, the images were analyzed and karyotypes were generated using a karyotype analyzer.

[0228] Obtaining and characterizing cell clones that produce trastuzumab

[0229] The expression plasmids of PuroBT+_Tras_Hc and PuroBT+_Tras_Lc trastuzumab (Tras) immunoglobulin (IgG) were used. Figure 11B CHO-M cells were co-transfected with the IgG-encoded vector and CRISPR-sgRNA to allow targeted integration of the IgG-encoded vector into the ERV109F integration locus. Cells resistant to puromycin were selected, and clones from single cells were isolated using a ClonePix FL imaging system from Molecular Devices, LLC.

[0230] CHO-M cell line and fed-batch culture

[0231] The parental Selexis CHO-M cells and derived clonal cell lines stably expressing human monoclonal IgG1 antibodies were cultured as follows: Seed cultures were passaged every 3 to 4 days before the N-1 generation. Four days before inoculation into the microbial reactor, CHO-M cultures were passaged in shake flasks at a cell density of 0.30 × 10⁶ cells / year. 6 Cells / ml (N-1), volume determined according to process requirements. Cell culture is conducted using BalanCDGrowth, supplemented with 6 mM L-glutamine (HyClone, USA). Culture medium (Irvine Scientific, USA), incubator set to 37.0℃, 5% CO2, and 120 rpm. (Germany)

[0232] Total protein quantification analysis

[0233] The automated microfluidic capillary gel electrophoresis system, LabChip LCGXII system (Perkin Elmer, Inc.), was used for total protein analysis. Protein-containing samples were mixed with amine-reactive fluorescent dyes that non-specifically labeled the proteins, and the proteins were detected at the exit of the separation channel using laser-induced fluorescence.

[0234] Clonal characterization of targeted integration efficacy

[0235] The genomic integration site of the Tras expression vector of IgG-producing cell clones was analyzed by q-PCR analysis of genomic DNA. Quantitative PCR (q-PCR) was performed in multiplex mode using three Taqman probes. Two Taqman analyses were designed to determine the presence of the ERV109F linker sequence between the ERV and genomic DNA on each side of the ERV integration locus on chromosome 15 (Chr). Absence of amplification indicated that one or more transgene copies had been integrated into the ERV109F allele and that the ERV sequence had been deleted. Figure 12A and Figure 12B A third Taqman analysis was designed to assess the presence of the WT allele, i.e., the ERV-deleted chromosome 9 genomic sequence, thereby evaluating transgene integration at that locus. Figure 12A and Figure 12C ).

[0236] If no products are obtained from any of the three q-PCR analyses, it can be inferred that the transgene copy has been integrated into both alleles. Figure 12AIf all three Taqman analyses produce positive results, this indicates that both alleles are intact, and therefore the Tras coding sequence has been integrated into the rest of the genome. Figure 12D Therefore, these clones are classified as clones where transgene integration occurs randomly.

[0237] ERV109F expression

[0238] According to the manufacturer's plan, use Qiagen. Total cellular RNA was extracted using the kit. DNAse treatment was performed twice, once during extraction and once after extraction. PROMEGA's [technology / method / technology] was used. The reverse transcriptase (RT) kit reverse transcribes RNA into DNA.

[0239] TaqMan analysis, designed to detect long terminal repeats in ERV109F, was used to perform RT-qPCR analysis on ERV109F RNA from Tras-producing clones and parental CHO-M cells, using cellular GAPDH housekeeping gene mRNA as a reference. The fold decrease in ERV109F expression was determined using the ΔΔCT calculation method (Livak and Schmittgen, Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCT Method, Methods 25, 402-408, 2001). This analysis allowed for the identification of reduced ERV expression following transgene integration at the ERV locus, further validating transgene integration and ERV sequence deletion at the ERV109F locus.

[0240] Analysis of the ability of cultured and cell clones to produce trastuzumab antibodies

[0241] The cell culture procedure used to determine Tras yield in fed-batch culture was performed as follows: Cell growth and production performance were assessed using classic fed-batch static cultures in 96-well or 24-well plates under stirring. A cooling system was provided to allow for temperature variations. Batch feeding cultures were conducted in an automated microbioreactor system (Sartorius Stedim, Germany). All cultures used 40% dissolved oxygen (DO), with stirring speeds ranging from 1000 rpm to 1400 rpm, temperature maintained at 36.5 °C, then reduced to 33.0 °C (time variation depending on inoculum density), and pH controlled at 6.90 ± 0.10. CO2 and 1 M carbonate were then used, varying the pH to 7.00 ± 0.20 (time variation depending on inoculum density).

[0242] Batch fed cultures were seeded in 24-well or 96-well batches at a target cell density of 300,000 cells / ml using culture volumes of 3 ml and 250 ml, respectively. Following the inoculation density procedure in Ambr15, the microbial reactor was started with 1.00 × 10⁻⁶ cells / ml in an initial working volume of 13 mL. 6 Seeding was performed at a target cell density of [number] cells / mL. Cell culture supplement 1 and cell culture supplement 2 were added to the culture on different days according to the seeding density schedule. Glucose solution (Sigma Aldrich, USA) was added daily according to the glucose concentration. Good cell viability and high levels of production were maintained as needed. Microbial reactor samples were collected daily for cell counting and viable cell density (VCD) assays. FLEX2 (Nova Biomedical, USA) measures cell viability. Cells have been grown for up to 14 days.

[0243] As those skilled in the art will understand, the above description is not limiting, but rather provides embodiments of certain implementations of the invention. With the guidance provided above, those skilled in the art can devise various alternatives not specifically described herein.

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[0276] H.Neurath and RLHill, In: The Proteins, Academic Press, New York (1979) sequence list <110> Silex Ltd. <120> Targeted integration to enhance gene expression in mammalian sequences <130> P100777WO / 3024-286-PCT <150> US 62 / 953,405 <151> 2019-12-24 <150> US 62 / 960,367 <151> 2020-01-13 <160> 60 <170> PatentIn version 3.5 <210> 1 <211> 59558 <212> DNA <213> Chinese hamster (Cricetulus griseus) <220> <221> misc_feature <222> (1)..(30020) <223> CHO genome outside of ERV on the 5' end: ERV-c 109F <220> <221> misc_feature <222> (30021)...(39247) <223> C-type ERV, LTR to LTR: ERV-C 109F <220> <221> misc_feature <222> (39248)..(59558) <223> CHO genome outside of ERV on 3' end: ERV-C 109F <400> 1 tggttctatc gagacagcta catccgcttc tccacacagc ccttctccct gaagaacctg 60 gacaagtgag ctttccctcc acctcccttg actgcccagg ctagtgagga ggtcagcaga 120 caaacagaac agtgggctct gcgggcaggg aaggcaggct tttcgccgcc actcatcctc 180 tgccctgaag gagcttgcca agagggtgcc cttgggttac cgaaatgagc aaaacaagaa 240 ctcctgctaa tcaagggctt acatcctagc aatgagcttg agtaatcatg gctaacatgc 300 tagaaggtga tatgtgttag gaagaaaatg tagatctggg taatggcaga ttaagagaaa 360 cttgagaggg gaaaggcaac ttgcagtttg ctcttgggat gagaggaaac cttgaggaga 420 cctgggttca aattctatct ctgcagcatc cagattgtgt tcctctagag acatgaaaag 480 agacttaagc ctcagtttca gttagagccc agtggtccca tctgtaatcc cagcacttgg 540 gagtctgagt ctgaaggatt ttgaggtcat tctgggctat ataatgagac ccccactcaa 600 gacaaaacaa gccaggtgtg gtagctccta actgtaatcc tagaacttgg ctaaggcagg 660 agccaaaatc aagccaggta cggtattgga ggcccgtgat cccagtgctt agaaggtaga 720 ggcaggacaa tcagttcact gtcaccctca gctacatgcc aagttttatc taacctgggc 780 aacatgagac catttcatag aaaaagacca gcctccgttt ccacatctga agactggagc 840 agcaatacac tgtagctgct tatcatcaag tacttcacat gtgataagtt gttcctgctt 900 ggttatacca cagccacttc tcaatggctc acaatgaagt gagttattgc cattttaaaa 960 agagatgaga ggggcaaagg ttaaatgact tgcctgagat ctcatagttg atcatcggtg 1020 aagctgggat ttgaccatcc agggtcatcg tccccctgcc acacatagga atggtgaact 1080 gaaactaggc ccaaagcact tgatacttac tgtggcctgc aggcaacact ggagcattct 1140 gagaaatgac tagccctggc cgtgggtgaa tagggagtgg ggcagggagt gggggtgggg 1200 tggaggggtg cagcttctgt gcttctgtcc tgttgatctt ccagcagggg cccctctcag 1260 ttgagatccc ctagacggct gaccacaggg ttctgcctgc agccctgagg ttgggctcca 1320 gggctgagcc tgtgtctctg atccactcta gctctgtgca cctatgtaac aattccatcc 1380 agagatacct ggagacctcc tgtcaccggc accggatgct gccctcggac aacatgtggt 1440 ccagccagag gtttcaggcc cacctgcagg aaatgggtgc cccaaatgcc tggtctagtg 1500 tcattgtacc cggcatgaag gctgctgtga tccatgccct gcagacctcc caagacactg 1560 tgcagtgccg aaaggccagc tttgagctct atggggccga ctttgtgttt ggggaagact 1620 tccggccctg gttgattgag atcaatgcca gccccaccat ggcaccttcc acagctgtaa 1680 ctgcccgcct gtgtgctggt gtgcaagcag ataccctccg tgtggtcatt gaccggcgac 1740 tggaccgtac ctgtgacacg ggagcctttg agctcatcta taagcaggtg agatgtccca 1800 gcacctccca caggcaaccc tacagcaaag ccctggctgg ggtctgctgt gagacagagt 1860 tcaagactga ctctacacac ggggcatctt aacacagaca cgtcccactg gcctgtctcc 1920 tcatctgtgg aagatactgt cctttgagag ccattaatgc catgagtttg tagtcatagg 1980 tagtattttc agaggccctg ggacctgctc agtttccatg gtaattccaa gcctctaggt 2040 agtaaccta ttctctcatc tgtaaagtaa gactgtacct ggctcctcct ttgtgtgctg 2100 tgagaatggg tggttgatc ttcacacaag ggtttgctaa agtaccaagc tggaggacat 2160 agaggatatg aggccatgaa cctcaaggcc tgcctatcaa gagttaatta ttagtgtcta 2220 tcattgtat aacgattatt atgttactca tcttcttcca aaaacagatt tgatcttgct 2280 tatttataat aagtatagaa ttgcttgggg tttttgtttt tgtttttgtg ggttttgttg 2340 agatagggtt tttctgtata gctttggatc ctgttctgga acttgctctg tagaccaggc 2400 tggccttgaa ctcaccttg actgcctctg cctcccgagt gctgggacta aaggtgtgca 2460 ccaccattgc ccgacctaga attgtttttt attcagccaa aaaacattac cttgccctcg 2520 tggtctaatt ctctctagaa acacccttag gagcacacca ggggcatgcc ctatagaaat 2580 cctaggtttt ctcagtccag tctagttgac aactgatatt agccaccaca gctggacatg 2640 gggctccacc ttccacctcc cagtatttgg aaggctgaga caggggatca cttttagttc 2700 aagggaagct tgggttacac agtgagttcc aggctagcct gggcttcaca gtgagaccct 2760 acttaaaaac atcatacaaa caaacaaaca aacaaacctt taaacgtatg tttttaaggg 2820 tttctgaatt ctgaggacag tttttaagat ctttgagcta agattcacca aggtctaggt 2880 ttgctacagg aagggaaaac actgatcacc tgacctgggt ggcctcatgt ttcctacagg 2940 tcctgatcac cttagaataa tgtgctggca aagggttccg tctttgttag ggatgccatc 3000 actaggtgtc cagggtggaa tccaggcctg cgtttttagc atgtgcagtt ctactgagct 3060 acacctccag cctaaaaaat gtaaaggagg agatgcatgc aggtgtgcat acacctgtaa 3120 tccctgggca acagaagcaa gaggactgtc acaggttcac caccacctgg ttacagggtt 3180 tataataagg tcctgtcaca aacaatgtag tcttggaaga gaggagagga aatggggaag 3240 aggaagtaat ttgcagttta aaatagagca aaattgccgg gcgttggtgg tgcacacctt 3300 taatcccagc actcgggagg cagaggcagg cgcatctctg tgaattcgag actagcatgg 3360 tctacaagag ctagtttcag gacagcctcc agagctacag agacaccctg tctcaaaaaa 3420 aaaaaaaaaa aaaaaaaaaa agcgaaattt gtatggacat ggcaacacgt gcctgtaatc 3480 ccaacactta gggggctgag gttggaggat caggagttca aagttatcct tggctgtatg 3540 tgagcttgaa gccaacctgg gcctacagca ctgtctgttc cacaatctgt ctttatctgt 3600 ttgtctcctt atgttgttca gctcggtctg ttctctgaat gtttgtctca gaacaaacaa 3660 aattgaatag ggctgggcat acgcactttt caaatgtcct ttgtccccca ggtatctttc 3720 attgctgatt tggttttgag cttgagggtc agtgtacaac aaggtggtta caatggtggc 3780 tttgtctgtc tctgatctcc tttatctagg acagtgccac tgctgtatcc ctggcaccct 3840 ggtcctttgc aggccaggca ggccagcctg caccaccgcc ccacactgtt ttatctgttt 3900 ttctccttat gttgttcaga tcggtctgtt ctctgaatgc cctgtaaact agaagatagg 3960 ctaacaagct gatggggttc agggttgaga tttttggcaa aaaacactca tgtgatgcta 4020 ggtacctcat gtgactgtca caaggcacaa ccaggaatct ctagttccca atgccgaatt 4080 tgaccttaga ctaaggtggc tgccaccaga gccacatcct cccctttgta gcttttttca 4140 tttttcttta cattatttat tgtgtttgag tttgtacatg tgtgtggtca cacatgccac 4200 agcacacatg tgggagtcag agggcaactt atgggagttg gttctctcct cccatcccat 4260 gggtcctggg gcttgaactc agcaagtacc ttataagcta tctcaatact gtttgcctcc 4320 aaaatgtatt actttgtgta gctgtccctt ttctgttgct aataacagaa tactacagac 4380 agtgtaagtt acaaagaaaa taggctcaat tgtatccatc ttttgctgcc catggcatga 4440 aaacacttgc atcccaacac aggccagagg tcgctttagt gtgaagcagg attgagtgca 4500 tgtctgcatg gctaagactc tctacttctc tggtttcctg atccctaggg ctctgggact 4560 ctagatcctc tggacccctt gataatagag ggagcttcct gtttctcaga agtgcctttt 4620 gaccatatat cccagaaact gattccatcc atctctgccg tgtgtcacta gtcactagat 4680 ggcgtcactg tcatctctca ctagtgtctg agatggcctt gtcttctctc ctgcccacag 4740 cctgctgtgg aggtgcccca gtacgtgggg atccggctca tggtggaggg ctctaccatc 4800 aagaagccca tagcagcttg tcatcggcgg acagcggtcc gctcatcact ccctcatctg 4860 ctggcccagc aaggctgtgg ggaaggcaag gactcaggac cccctatcca caggtcagct 4920 tctaggaaag atgctggggc caggagcctg ggacacactg agaagccaga ctctgcggcc 4980 accacctcag tccccggaaa ggggaagaaa ggcaaggcaa aaagtgccac agccctggtc 5040 tgcatcaccc tgcagaaatg ggagtcccac aacaccaggg tgggccccac cttcaacagg 5100 ttaatgtgtc tgaaacagcc tgaggcctgg ggtagtacca tgtcccccaa accccgcagt 5160 gttcccaagg ccatttctgc ctgctctcca agccctcccc aagcatctgg gcttgccctc 5220 ctgccaaaag gccaccagtg atagcaagta tgaaccaaat atctttaaat acataaccaa 5280 atgagtatta caaagtagtc accctgccag gcagttagac caaaggctcg gtcctagagt 5340 gcgcgcccag agtccagacc catgctgctg ctctagccag cctttgccct cacctttctc 5400 tggagaaagg tgctgccacc atgcccttcc ccattcctaa ccagccccct cagccctcat 5460 aacgccctag tgaggtaggt gctattgtcc ccattttcca gccgaggtag cagcaagttt 5520 aaggacgttg cccgaggttg cacagctcag aaggggcaga gctgggatgc agacccaggt 5580 ctgttggtct cccaaccctg tgttcttccc actgcctctg gaggaggagc tgggaggggc 5640 tccatctgcc cttaccttgt atcccccacc tttacacatg tactgtggaa caattggtca 5700 ggctggggcc tcacccagat cctcacagct tcccttctcc cacagccccg tcctacctcc 5760 ctagtctcca ttccaaggcc tggctgcctt cttcccatgt gctccgaccc cagggccggg 5820 tcctcagact accgaatggc caactggtgg gctctaaggc tctgtcaacc acaggcaagg 5880 ccttgatgac tctacctact gccaaggttc tgatgtcctt cccacctcac cctgatctca 5940 agctggcacc cagcatgctg aagccaggaa aggtgggcct cgagctgtgc ctcacaccct 6000 ggcgggtagt gctgagcagt gggatcgggg ctgaagggca cgaacagagg gcagcgctcg 6060 gaccatacag cgccccaggg aagggcttgt cttctccaga accctgttcc aagacagagg 6120 cctgatcata tctctttccc tcccctcctt gcaccgaggc tgctattccc ctgcaccttc 6180 gaggccccca ctttggaagt gcctcgaggc ctctgccctt tgaagttgga cctcttccta 6240 gcaccacag gaaagtcacg gccaaggca agttcaaggc catactctgc gawaagcca 6300 gggctgaggc attackccaag aagaggctga gcctccccaa acccttgacc cttattctga 6360 catgccggac actgagaacc atggggta ggaggctaga gaaacccctg ctctgatctc 6420 tactgcccca tcctggatcc agcatcaat taaaaaagc aattaaagtt ctctggactt 6480 ggcttgaata atgtgcggct aggctcataa aagagttgac cagcagggcc tccatcagca 6540 agggccacag tcccaccca gcgacagaca ttggctttct ctgcagggag acggatggtt 6600 ggggaaagag ccttcactat acgatgatg acactgagac atggcttgcc tgagaccaca 6660 gcaggcggaa agtcagccat caggagtgcc ccttcccaa vakaagctgg gctggcagaa 6720 gagcttctga tggtcacaga acatgac agagacggg ctttccctaa acctcagcct 6780 ttctccggag agtatgtccc tcagtgaggg gtcggtcaag acacctcaa ctgcaaacc 6840 CAagaaacag gtcaagtgtg gcattccta cctgtagagc ctcagctgct ggtcctccaa 6900 aagcggtatc tagctgtagc gtgtgaatgc ttcctgagtg gggcaggggtg gagaggagag 6960 tgcggtgttg aaatccaatg acctgtgtct cccaaagtca gaagagctca tgcctgcaca 7020 gtggtgtgtg tctgtcctcc caggacttgg gaggcagagg caggtgcatc tctgaattcc 7080 agtccagcca gggatatacc aagaggccca gcctcaaaag caaacaaacc tcctcgtgac 7140 caggagatca gcagatgcca cctccagacc tggccactgt tcacttgagc agagagcaca 7200 gtccctggtc aacacttgct ttctcagcag atcctcaaaa gcagacttgt gagtaggac 7260 ttttattatt ttaagtccag agagcggtca cctgcccaac gccacacagt aagtgagtgg 7320 tttaactggg atttatctta ggttggtagg actaagggt caaagacctt gaccgtactc 7380 agcaccaagc ccactgtcct tgagctgggt cacggccctt ctttctattt tccaattggg 7440 ggttaagcac tgtctatcgg tgagagagcc gcaggcactg cagggtccga gagagatcg 7500 aggtagggg tgccacaagt tcactagggg ggtccctgga tctggtgcct gggaggagga 7560 ggcggtgcaa gtgcaggtgc aagggcatct gggccacctg ggaggacgc aggcgaaggc 7620 gtctgaggag agcttcgtcc agcacctgga gtgggaaaga cagcagccca cctgagttcc 7680 agccagagga gcccctggct ctgatggacc tctctggtct gcaactgcca tcattttctc 7740 aacaggcagg cagggatttc tctccacaca gagctaagtt acgtttcagc tccttttgtg 7800 tttagtgaag ccatgtgatt aagccactca ccaatgggat gtgaggaaaa cacagaccta 7860 gccctcaaag ccccgattca caaaaacatc cagtctcccc ttatggccag gaagcaatag 7920 cccttttctc caagtggcta cccagagtca ggtccactcc tactgtatgc ccatttgcca 7980 gacttaatcc aagaagaaac aatggacttc agggcctgtc agaggtcagc tccccactcc 8040 ttgagctaac agacagaagg aagcctgagg aagtcacagc accacagagg caagggtggc 8100 cccagaggcc aagcctgttc ccccaattcc ctaaatacag agcaggcatg tgggcctgaa 8160 gacctacctg tctctggagc ttggtggtct tggctggcca cagaaagcgc tggcccagaa 8220 cggtgcccac tcgaggagca taggtgtggt ccccaagcac tgggcagagc tgtagagcca 8280 tgtgcacctg aagctgactg gggaacactg aaagatgggg ggaggcagtt gctcttcttg 8340 atcatcaagg actgtcccca ttcccagagc ctgtgcatct atgaggtgac ctctgcctcc 8400 acagagggcc atgagcagat caggacaatg acagctggca accttcccag cctgggggca 8460 ttgaacccaa cagaactgaa gctcctggga ataagtatgt gggttgggtg ctaaactgtg 8520 ggtgtgcacc taaaactctg cctcctgctg agcccactct cagagtcccc agatgctctt 8580 ttttttttgt tttgtttttt gttttttcag gcggggtttc tctgtggctt tggaggctgt 8640 cctggaacta gctcttgtag accaggctgg tctcgaactc agagagaggt gcctgcctct 8700 gcctccccag tgctgggatt aaaggcatgc cccaccaccg cccggccacc agatgctctt 8760 tggatgttgc aaaacaacat cttcctcata ggcttcattt cccctgcaga gtactgttca 8820 gccctacctg tcagtggctg cagctggacc agagcacagc cacagcctgt ggccatcaca 8880 tggaaatggc tgagggtcct cttgacacct tctaggatgt cctttcgaga tggggatgtc 8940 acggggatgg cctaggatac cagtcaagaa tgacttagca cacacgtagg agcccaattg 9000 agccacccct gctgccggct gacttaggac agggcctcag aacaggcagc agcaacttgc 9060 ctgttagagg acaggggaat agccacccca tgcacatagt gcagactcct ctgtaaggca 9120 aaacctgaaa ggactcctag tggcttctga ctcacaagat caatgccatc catgcgttcc 9180 agcttcaggg ccacgtggat agtcccctca gaaggctcag ggatgccatc agtgatgcca 9240 ctgagaaaga gaaaggaagt ctcagagcag ccagctggga ccttccttgg ccctgggagt 9300 caccccgcat ctctcaccag taggtggctg tgggcctctg tgttctcctt gagtgaacga 9360 agaacttctg caagcggctt gctgtctggg ggcagctgga gagaagcaca agcccagacg 9420 cctccctgaa agaagaggac aagtcacata aagcctcttg gttggaaaaa atgagggcca 9480 gattggctgc tcctgcagag gactagcact cggcacccac atagtgggtc ccaactgtaa 9540 ttccacttcc aagagagctg atgccctctt ctggcctctg gggcaccggg catgcactgt 9600 ggtacacaga cacacatgca ggctggcctc cgggggcaca aggcatgtgt ggtgcacgga 9660 catacatgca ggctaaacac acatttaaaa acaaatcttt ggtcttttttt aaaggagacc 9720 ctcccaccaa ggggctggag aaatgaatca gtggttaaga gcactagctg ctcttccaga 9780 gtacctgggt tcagcaccca catggcagct cacaactgta gctccagttc caggggatct 9840 gagaccctca cacagacaca catgcaggta aaacaccaat gcactatata tatatatata 9900 taggataga tagataga tagataga gagagaga gagaccac cttcccacct 9960 ccccaaatga acaccaggac tacagtccag acctaagaaa caaatcctgg ccaggcagtg 10020 gtggtgcacg cctttaatcc cagcacttgg gaggcagagg caggcgcatc tctgtgagtt 10080 cgagaccaac ttggtctaca agagctagtt ccagggcagc ctccaaagct acagagaaac 10140 cctgtctcaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaagggggg 10200 ggaataaac aaatcccaag accttttttc tggcccccag agactcaga CAATGCCct 10260 aagagttact accatgcca ggcacctgaa taggactt atacagtg ggtgtaaaat 10320 aaagcccac cacactccctc acttacttc caggtgctcg cacaacctgg agctcccggt 10380 gtttgagcccc cagggcctgg ctcagctgtg gcagcacaga aagcaggtc agctccccag 10440 gtctccctga gaagtaagg ggagaaaaca agttgccaga gcaccatcca acgaactcct 10500 ccccaccagc ctttccctcc tgtccctgc ccatacctgt cacaggcaga ccctgtggct 10560 tgttcagtgt caccagaggt cctgaaatat tcggtagatg tcagcaaaag cagggcagcc 10620 tgtgtgcatg cagcctctgg ttataagctc ccaagcccac tccaaactga acttcctgct 10680 ccccgcaccc agggcctttg cacctgctgc cctgagcttt cattaatatt gggtccctta 10740 tcacacacct ccctataatg tgtgcccctc ctacagtcct tgactgtttg gtctgagacc 10800 agctcaaaca ggccggcctc aaactcaact ataggtaagt ctggtcttga actcttgatc 10860 ctccttgcct ctgtctccca agttctggga ttacaggtgt ggtcaccact cccagaaaca 10920 gcagagattc actattcctc acatcagaac gcttgctctc tcaaggcagg agccagccat 10980 atttatccca gtactgtgta gcctcctcat tgctacaatg cactgaagtt cagcaaagtt 11040 gtcattccta aaggcacaca gcatgctgct tacagacccc aggaaactaa cttctgctcc 11100 cgagggtctt agaatgcgag gcggtgtgaa ggtttcctat gcctggcgga gggaatgtga 11160 cggctacagg ggtccgactc cagggggtcg gaggctccct caccttgctg atccaccaca 11220 gctgccctca gcacctcagc cagctcctcc gggccgaggt tctctgttcg cagaagcccg 11280 gggaagggct ggtcctccac tgcgtccttg cacttgctgg aaccttgagg ttgaggccga 11340 cccctgagaa aaacgacaga agccggtacc tccaacgccc acgctgccca gtgcagcctt 11400 ttcgtacaca accctcagaa cccccggctc ggctgacagt tgtggtccac aggcccccaa 11460 gactacagaa ggggcatcag acgctcggag cgccaaccat ctcgtgtcgg gtcacacaac 11520 gcggctggga ttagaaccca tgtcctgatc tgagtgcccg acctgccctc cccttgtcct 11580 agcctccctc ctacacggaa gtgttgagtt ataatcaccg ggcctcggtg ccgaagcctg 11640 catctctcgg ccgtgcgcca gccgccagcc ggggccgcca ggtgccagac acacaacgcc 11700 aaacgcggaa gacgcccatc gccgcagcaa gcacagacgc atgcgtgtcc acggactgcc 11760 ccaccgcgtg catcctccgt gcgccgattg gtcagcctgg ctgtcaatca gagcatcgag 11820 caggcggagc ttcgaggacg gaagagccaa actttccttt ggctgaggaa ggtaaagtac 11880 ggtctccggc tctgctttgg gggaaactga ggcaggaggc gcggttattt actcgcggta 11940 gaggacgtgt cttagaatag aaagaggcga gtttgcagat agattttc agttctcagg 12000 acccttaatc tgaaacccat ccttgtaaag cagagggaga aggtgaagcc ccacagcctt 12060 cgggccaggg cacctgcatg gaatggtct gcgacattaa ccatcccatg actgtggctc 12120 aggtggtaga gagatttttc taggatgcac gagttcctgg gttcgatccc cagcaccaca 12180 cgaagaccaa gatcacagat acagactgag ttggagacca gcctgggata tgtaaggccc 12240 ttctatcaca agaaaaaaaa actaggccgg gtgtgtggt gctctctc aacagaggcc 12300 aacctggtct agacagtgag ttcaggaca gccggagcta cacagagaaa cctgtcccc 12360 cccccccaaa aaaaaaagaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa ur lav la la la la ver back the higher the longer the longer the longer the other, the higher the other 12420 cactggccag tatgttgtta attccaaaga tggaatgagt gtaggaaaa aggccagcca 12480 aagaaacaca ctttggctct gcaaccagaa ccaagaat gcaccttgt cagaagccag 12540 ccttggtgac gcccccccc ccaatgccta gctagcccc agctagccta gcatcccacc 12600 cctcaggttc caggacctgg cctgagaagc aactaacacc ttcccccatt tccttagtta 12660 12720 tctttctcca cacaggagat tccggatacc tgcctgagag caattaacat tcattcctcc 12780 cccacctcct tttctctgc ttcctccttt ctctataaaa accccttgta ataatcaata 12840 aatgggcctt gacaagaaaa cctgcttggt cccgctcttt ctttcccgcc catcattttc 12900 aggcgtgatc cacctcggac tcaggaatta ccggagcccc gagggccggg gtacaccctg 12960 accctgaaac cagtgccaga gaacacact ttggccctgg aatcagagcc agtgaaagaa 13020 atacaccctg gctctaaaaat cacagtcaaa aggctaaaaa ggaccagtga aaaggaacaca 13080 ctttggccct gaaaccagag ccaaatctaa ccctaaacct gtgcagaaaa ccaaccaatc 13140 cctgagcagg agatctagcc attctgagct cagggattga aatctgacca atccccaccc 13200 tggaaatctc cctgggaaaa ctctgccccc ccctaagaat ccctatataa accctgtgcc 13260 tgttcagctt caggctgcct ggctgccctc tgccactgga ttcttgaatg aggtttgggt 13320 aagaactttc gccggagcag agaccaccct acacactggc tctccagggg actggagttg 13380 aactgttaca atttaaccag ggaaaccctc tcctccccaa gcagagctga tacactgagg 13440 aaggcctttc cctgaagcag ggctgtaagc cgctatgctt actgtgacct gtggcattcc 13500 ttggctccta aacgccagaa tacctttcca tcccagctgt aacactcagt attctgtgac 13560 tccggagtgc cagaatactt tgcatctgag ccataacaca gtattctttg gctcccgagt 13620 accagaatcc attccttccc atccaatccg ttgaaatgct tacaatgaga aaaaccacga 13680 acccaaatca ttgagaccaa attaattaaa gttttttttt cattcatgta cgagactgcc 13740 tcccccaagg cctgatttga gaggtcagca ttggatgtga ggaagacaag ggggttttgt 13800 tgttttggtt ttttgagaca gggtttctct gtgtagcttt ggagcctgtc ctggaacttg 13860 ccctgtagat taggttggcc tagaactctc agagatctgc ctgcctctgc cacttgagtg 13920 ctaggattaa aggcatgtgc caccaccgcc tggcctcacc tggatcacac agacacatat 13980 atacataatt aataaaatac agacctggcc tttaatccca gcactaatct acatagtgag 14040 ttgcaggaca accagagtta catgaccctg tctcaaaaaa aaaaaaaaa aaaaaaaaa 14100 acaacctaaa taaataaaat gcagaaagta acaatatgta cataataaaa ataatgaaat 14160 aataacataa ttgaggaaca gtggggcatg gtggcatgtg cctttaatcc catcacttac 14220 tgatctacat cccaagcctg aaggaccttg ctgaaagggc tccagcacta gactgttgct 14280 tctggcagag aaagtggctc tcaacctgct ggtcaacgac ccctttgggg gtagaagggc 14340 cctttcacag gggtagccta agaccatcag aaaatacaga tattcacatt acaattcata 14400 acagtagcaa aattacagtt ataaggtagc aaaaaata attctatggt taggagtggg 14460 tctccataac atgaggaact gtattaaagg ttgcagcatt aggaaggttg agaaccacca 14520 atttaggggc agatgctgcc tccttcacag cactcagcca cagttgtgtt gtgtccttgt 14580 tgaggttcta tccggtcctc caaagttcag ctgtgggaaa ctaaatcttt aacttcataa 14640 taatggtaat tggaggctgg agtaattagt accagataaa atcatgaaga gtgtgcttcc 14700 atgatggcca ttaacagctt tctaaggaga gcagagaaaa aaaccttttc ctcgttgtgg 14760 aatgacttcc tccgtgttgg tgtgcaccag aaaggccccc tgcttgtgct aagtggagct 14820 atgccatgct cttggactct gcagcctcta gaaccatgat ccagatcaac tgtgtgtctg 14880 tgtgatttgt tttattttta ttatgtgtgc atgcctgtgt gtctgcgtaa gtgtatacca 14940 tatgtgttgc aggtgcacgg agaggccata aaaggggcat cagatcctcc gaagcttagag 15000 ttttaggcag tcgagagcct ccacgtgggt gctgggaact gaactcagag cctctgcaag 15060 ctcagccagt gctcagccag tgctcctcag tttgtgtgct tgttttcagt actgcagatg 15120 gatccaggac cccacacgtg ccaggcaagc attctctcac tgagctgcac cctcagactg 15180 gtctcaggtg gtttgtata gcaacagaaa acagacatgt ggaaggagtg atggcgtatc 15240 ttgactgaga ggaggaataa cttggctact cgcacagcac atgtctgggc aggttcacaa 15300 ggaggcttcc agggacaatt ggcacctaag aaccctgacc taatgaatgg atgaaccatt 15360 cagaatttgg atgggttctt gggggggggt gagggaactg tggtgggtgg ggactgcttg 15420 gaggaagtag ggtgtttgtt gtgggtggct ctcggaggct atgtcttaca ttggctcctt 15480 tctgttttgg ttctttcaat ttcctgttgt gtgctattct gctacaaccg cctcacccag 15540 tagattgcag cctctgttaa tcaagacagg gtcttactgt gtagccctgg ctggcctgga 15600 actcatagag atctgtttct tctgcctctt gagtgctgga attaaagacg tgtgccacca 15660 ccaccacctg acctagagga caactttatt ttttttaatt ttttatttat tatgtacaca 15720 ttgttcctgc ctgcgcacca gaagagggca ccagatctca ttatacatgg ctgtgagcca 15780 ccatgtgggt gctggtactt gaactcagga cctctggaag agcagccggt gctcttaacc 15840 tctgagccat ctctccagcc ccctggatga ttaattgtag aagtttcagt tctttctttt 15900 tgtttttgtt ttttgagaca gagtcacact acgtatcttc aactgatctg gaacatgcta 15960 cgtagacaag gctggcttca aatgtgtgtc aatgttcctg cctctgcttc cagagtgctg 16020 ggattatagg catgtcccta aatctgttat ttttttaaac acttattttt gagggggagt 16080 cggtggtggt gctgttgtac gtacatatgt agagatcaaa gggcatcttg tgggagtctg 16140 ttctctcctt ccaccaatgg gttctaggga ctgaattcag gttctggagc ttgacagcaa 16200 gcaccttaat ctgctacaac atcttgttgg tactaaatcc atggagaatt gaaaggattg 16260 tttattgttg ggcccgggaa tatagctctg tggtagtaga atgcttgcct aatatgtgtg 16320 agattcttaa aaaatcattg attgttttct taaaccaact ttttaatttg ttaaaaccaa 16380 cttttaaagg atatatctag gcatggtggc acacagcatt aaccctagca cttgagagat 16440 agaggcaggc agatctccta tgaatttgag gccatcctat tctacgagtt ccagtacagc 16500 caggactaca ggactgtgtg atatgcacaa cacaggaagg actgatgaca aatttggtaa 16560 aaaaaagaaa gaaaaacagc aagtggtttc acattgtccg gcttcacctg gagtgggtga 16620 ctgtgggtac tcctgaagcc tcccaggagt gggtgactat tctgtcagac tgtgggtatt 16680 cccgaagcct cctaggaggc ctcctgtgca tcacagaagt ggcttgagca agaacttggg 16740 acagactggg caagtgcaca gctataatcc cagcactgca gaggtgaggg ctgatgggtc 16800 aggagctcac agttatcctt gactacagga agtctgaggc cagtctgagc tatgtgagac 16860 tctaaaacat acatgagtca gtgagttggc tcagccagta agggtgcttg ataccaagtc 16920 tgaccacctg agtctgatcc ccagaatcca catggtagga gcagagaacc aactcccagg 16980 agctgtcctt tgacctctgg acttatgcta ttgcatatgc ataccaatac ataactccct 17040 aaaatacaaa atacatatgt ttgggctttt tgtttgtttt gttttgtttt ttcgagacag 17100 gttctctgt gtagctttgg agcctatcct ggcactcgct ctggacacca ggctggcctc 17160 caactcacag agatctgcct gcctctgcct cccgagtgct gggattaaag gcgtgtgcca 17220 ccaacgcccg actgatgttt gggcttttt gatggtttt atgtgtgtgt gtgtgtgtgt 17280 gtgtgtgtgt gtgtgtaatt agaaggagaa agagagagag ggagagagag acagagagac 17340 agagacagag acagagacag agacagagac agagagagga gagagagcca aagtctggtt 17400 tgtggcccat tcaactaagc agacttctcc tccaccaatg gtgtccacca atggcccagg 17460 aaaggccatt tccccacttg aaccagcgcc tggccctctg gagggttagt tcctttactc 17520 tttggaggtc tccctccctg ccccatagca ggctgccctc tcttgctccc ttcccacagg 17580 cagctacctt catcccagtg ggctatgagc tcctggaaga aaaggaggag ccaaattttg 17640 cctaggctga gggctcagtg ccaggtggca gccacagtca actgctgaac ttgtgaaact 17700 ggaccccagg agaagaagcc tgggacaaac atcagcttgc atcagctccc tctggctcag 17760 ttacagcttt gcagcaggtg tggacaggct ggtgcttcag caatgggaaa caggactgat 17820 gcaaaccagg cctccaggag gccaagccct ggagccagcc tgcagtgaac cagcctccag 17880 ccacatctac aactgtgtga gggcctagaa tagaaagtgt acttgacctg tctgtccttg 17940 agtgctcttc tcttctcaca cttggctcca ggcatggggc agcttccagc gatggctttc 18000 cagacaagct tgagtaactt ggccctttgt cttagtgttt tctaaatcca aataggcttt 18060 acagggagga ttgtgaaatt atgttagctt tggatctgag agccagactg aaacctggct 18120 tcctacaggc ctagtctttc ctgcttaggt cagttacctg tcactaataa atgggggctc 18180 cttttcctac cagccaccaa gatggctgaa gtggaggaga agaaatgaac cttccacaaa 18240 ttcacatact acaatgtgga cctggatcag ctgttgacag gttctaggaa ctgctgatgc 18300 agttgtacag caccccagag gtggtgtctg aaccacggcc tgtggcagaa gcagtactcg 18360 ctactcaaac acctgagaaa gctcaagaag gaggcaccaa ccatggagta acccgaggtg 18420 ctgaagaccc acctgaggga cacgatcatc ctgcctgaga tggtgtgtgt gtacaatggc 18480 aaaaccttca gccaggtgga aatcaaacca gagctgatca actgctacct aggcgagctc 18540 ttcatcacct ccaagcccat gaagcatggc cagcctggta ttggtgccac ccactcctcc 18600 agctgcatcc ccctcaagta gctgtggcca acaaagactc atgtttaaaa agaaaattgg 18660 aagccaggca ttggtggcac acgcctttaa tcccagcact cgggagacag aggcaggtgg 18720 atctctgtga gttggaggcc agcctggtct ccagagcgag tgccaggata ggctccaaag 18780 ctacacagag aaaccctgtc tcgaaaaacc aaaaataaat aaataaataa ataaataaaa 18840 taaagaaaag aaaagaaaag aaaaatgggg gctcactctg agaagacctg actcctcctc 18900 ctcctcagaa ctctctggtt tgtttttgca gtgctggggt cactgtgcca ggctagagac 18960 tcactgcaat ctgagggtgg aagtgctgag caggaacagg gaactgtgta taagctggca 19020 taggcattga taaattcccc tgtagatgga ccaggacctt tcaacccgaa cacatggaaa 19080 gttattgtaa aatacaacag ttgggaatca aaagagtggt ttgatccacc ttacaggcaa 19140 atttcattcg gaaactgaca acacatatgg atcatgtggt ctgcctgaat atcaaatatg 19200 gcaggcctca aaaatcaact gcagatttta atataacatt taattattca tctattaatt 19260 aatttattct gtctcataat atcaaacctt atctattaaa agggagcagg gctggggatg 19320 tggctcagtt ggtagagaat ttgcctagca tgctggaaac cctgggttcc gggttcaatc 19380 cccagagcca cataaattgg atgtggtgtt tcacatctgt aatgctagca cttaggaagt 19440 ggaaacaaat ggatcataag ttcaaggtca tcctccacta cataataaat ttggagccag 19500 cctaggcttc tgtatctaga agaaaaaaca gggcaccact gatgcaactc attgcataaa 19560 agcaattgct gtgtgagcct gacaatccaa gctcaatcct tagaaccaag agtggaatga 19620 aagttgtctt ctgggccatg cacgccttg atcccagcac tcgggaggca gaggcaagtg 19680 gatctctgtg agttcgaggc cagcctggtc tacagagtga gttccaggat aggctccaaa 19740 gctacacaga gaaaccctgt ctcaaaaaaa aaaaaaaaa aaaccaacaa aaaaaagaag 19800 gttgtcctct gacctccaca cctgcaccat agtactggca taccaacaca cacacacaca 19860 cacacaca cacacacacc agtaatgaca aatgctatct cctgtaattc tcggcaaata 19920 gtttcaaaca aagaacacag gcaaagatga ggtattggca taaataattt acttcaggct 19980 ctaataccat acattagtgg ggaatgagaa caaaaggagg aattgtctga cttcccctgg 20040 ggatcacaaa ctctatccat tcggcccagc aagggcgcca atgaaaggtg aagaagccac 20100 gattccatcc aagtccagct tggtgaaccg gtgagtttac taggttactt acaggtgggg 20160 cctgggtgac tcaaaatcac aggtgactcc ctccaaatct gcatcagtga catcctggct 20220 ttagttaacc ttttacctct fatherctct cccgccc caagatcatg cccggg gcggggcagg agggagat ggctgggatt tcaggtgcta agaccccctg acactctcct 20340. ccctttctaa tacaggtgtt aactatttcc ataccctagc cataggcctc accgtcactg tggcatttgg ttcattttgt tgtcttgatt caggtcaaag tattctggag gccaccatag 20460 caatgtctgt tgcttgatga gcatggtcaa ggcaggaggg gactgcagag agggagtggc 20520 acatagggat ggcatgtgaa gaccgagtgg cagacttggt gccaaagcct gccagggaca 20580. agtgtttgtc ccctaaacta cctgtgacat gaggag actgagccag gctaggaagt tctcccgtga ccagcccacc ccagagctcc agcccttcct gcagtttcct tggtgctgtg 20700. ctgtgagagg ggtgcaggtt ggtgagaagc tctccagctg ccaggcttgt gggtgcttct 20760 agcagagtgc aaggtctcca gtcacatcct tggctgggga cggcatctga ggacttgggc 20820 cttcattgca gcatcttcag acaggcgggg aggagggagg aggtcttggc cttggacggc 20880 tgttcttcct cagtggcatc caggaactgc tgcatataac tggaggagcc aagcagcatg 20940 tggcctgtgg cctgcatgct gaagagggcc cagcggagca tttccctggg agacagcaag 21000 gcactcaggt taaacactcc ccatgctatc tccccaaatg tcttctccat ttttctatgc 21060 tggcctaaag cagttggctt caagttagac ttctttcttt gtttctactt cttttatttt 21120 tgagatagaa tcttaacact ttatctcagg ttagctagaa attcatttat gtagctcagt 21180 ctggctttga actcacagaa atctcctgcc tcaatctcct gatgctgttg cagttatggg 21240 ccaccacacc taactttttt tggtttggtt tggttttgat ttggctcttt ttgaaacaga 21300 atctggagat cctcctgtct tgtcctccca agtgctaggt atacagacat gtcccaccat 21360 actgggctca agctagcttt tctccttagg aagatttaag tgtcactaaa gtaaaatgac 21420 aaaaatgctt ccaagtacag acaggaactg ggaaccaggc tcatagctag ccatccctta 21480 cacatagttc tgcctctact cttgtgacaa cttttctgga accccttgct ttttcccatg 21540 cttaacttga gttctcaaaa caaagctttt tattttcttg taatgatatt ttgtttcttt 21600 cttttctctt ctatggtgct ggggatggaa cccagggctt tatttgtgca taagcagctg 21660 acctgccatg gagctatgtc cccagcccca tatatttttt ttttcaagac agggtttctc 21720 tgtgtagctt tggagcctat cctggcactc actctggaga ctaggctggc ctcaaactca 21780 cagagatccg cctgcctctg cctcccgagt gctgggatta aaggcgtgtg ccaccaacgc 21840 tcggcatcca gccccatatt ttttaaacta ctctggacca gccagattaa tttacataac 21900 acatgtctac cctttgtaac actgcttctt agactttata gtcttcccag gccagttctc 21960 agaatgctgg ccacaaggct cccatgcctg atatcatatt ctaagcatag aacttggacc 22020 agacagggct gaacactgat ccagagtggt ttgtttatat ctaaaatatt taaaatatat 22080 tttaaaatat tataaagatg ggtgatgtaa ctaatttagt gcttgccttt catgaacaaa 22140 gccctgggt tgatccccag caccgcaata acccagagtg gtaatatagg actgtaaacc 22200 taggatccag cactgtggag gtagaagcag gtggatccca agttcaaagt catccttggc 22260 tacatagcaa gtgtgagacc agcctgagat acatgagacc ctgccaaaa aaaaaaaaaa 22320 aagctttaac tgttctggtt ctgagctcca gcagccagaa gctttgtgac ttgtaaaatg 22380 ggtaatgagt ttgaactaa tgtgaagcac ttagcattg gtctgatgga cagaaaatgg 22440 gatagcagc aagagcagc tggctgaatg caactctctcc cacagctatt gatagggct 22500 cggcaggggt cactaggagt tgctggtctt aagaacaata aggagaa aacacagaaa 22560 agagaaatac tgccaggccg cggtggcaca tgctttaat cccagcactc aggaagtaga 22620 gabaggcaaa tctctgtgag tcgaggcca gcctagtctt cagagcaagt tccaggacag 22680 ctacggttac atagtgatac cctgtctcaa aaagcatat atatatatat atatatat 22740 atatatat atatattgaa tttagtggcc tgaggcaatg gctcagtggg taaagtgctt 22800 gccatatggc catgaggccc tgaagccca tgtaaaactg gggatggctg cctgtatctg 22860 tgaccccagt actcctctca tggtgagaca gagacacaag agactcctct gaagctttca 22920 ggccagcaag cctggcccag aacagaca aacagcaaag accctgcttg aaacacagtg 22980 gaagatgaga ccagcacctt aggctgtcct ctgactcga aatgcacgct gtggtacatg 23040 ggtgcccaca ttcacaca tatagagata aagactggct gggcagtggt vakacacgcc 23100 tttaatgcca gcacccttga ggcagaggca gttagatctc tgagtccaag gccagcctgg 23160 tctacagagt gagtttcaag acagccaggg cacacagag aaatagaaa aggaaaaaa 23220 aaagatata aagactgaa ctgaaaataat atattag ggctagagt atagcttaat 23280 ggcatggtgc tgtctagca tgtatgaagg tcctggttta atccccagct tgaagcatg 23340 tgtgtgtgtt tgttctttt tgtgtactg gggaactgga cacaagcct taggcagtg 23400 ctctgccatt gaactacagc ctcagctttc ttttcttt taaaaatgtt tattatttt 23460 atgtatatga gtactctatc tgcatgtatg actttatgct agaagaggc gtgagatccc 23520 actatagatg gttgtgagct accatgtggg tgctgggaat tgaactcagg acctcaggaa 23580 cagcagccag tgctcttaac cgttgagcca tctctccagc cccctcagct ttcttttac 23640 ccccccacac acaatatctc tctaagttgc ccagtttagc cttgaactta ctctgtagcc 23700 taggcaagct tctaatttgc catcctcctg tctcaatttc ctaggcacct gggagtacaa 23760 ggccatgtat agctttatat atatgttcg tgcagtgcct tcaaagttat ttctcaaatt 23820 agagaactga gttttgactc tagccagcca gtcttaaaaa ggaatgaaaa taaggcctat 23880 tctacaaatg aatgaacctt gaaaacagaa ttcttgtggg gaaagaatca tgatcccatt 23940 tctatgaggt ctctaggata gatcaatgga gcaacagaaa gtagagtaaa ggtgagcagg 24000 gtctcgggag agggctgaga accattattt actgagtaca gcttctgctg cctggtgcaa 24060 aggttctgga aaccaaggca atgcttgcac aacatgatta gttacttcat gctagacact 24120 tcaagcggcc acagttgaaa aatgttgaat gtgcagattt tagcacgggt gttttttctt 24180 gaaggtctca ttgtgtagct ctagctagaa tttggaactt gaaatgtaga ccaggctagc 24240 ctaaaattct caggagatct acctgcctct gcctcctgaa tgctggctgg gattaaggga 24300 gtgagctacc acacctgacc ccttagtaca tttttttttt taaatcacag taacaccact 24360 24420. aagtcactca gctaaccaca gtttgacatc agtttttatt ttctctaagt ttttttttt gttgttgttt tattttgttt ttcaagacag ggtttctctg tgtaacagcc ctagctaccc 24480. tggaacttgg tctgtaacc agactggcct tgaactcaca gagactcacc tgcctcagcc tcccgagtgc tgggaccaaa gatatgactc ctggcctttt tattttcttt ccatgtgtgg gaggtagggg ttatgcacct gagtgcagtg cccacagtgg tcagaagagg gcaacatatc tcctagtt gcagttacat gtggttgtga gctggctgag gttggtgccg ggaactggac 24720 ctaggtcctc gggctgagtg gttccctttt tctgtttttg ttttcaatac agggtctgat 24780. ctggcccagg ctgtccttga actcctgatt ctacacctcc aaagtactgg aattatagtt 24840 acattttcac ttacaaaaat attagctgtg gaaatagctc attattgag tatgtgctta gcatgcgtgc tgccctgggt cctatcccca gcacacaaaa gaaagcacat gactgtcatt ctttgttagc ccccagtgcc aacccaggct ttgagagagt tcagctttgg gtagacacaa aggacctctg gttagctagt ctcccaccct gctgtcccaa tctactcact tcacgacgcg 25080 cttggcccgg tagcagtgca ggtcatagga aagtgtgtat gtgtcataga gggtcgcctt 25140 cacgttcagg cagccgatga agtcctgggg gttcagcttg tataggtcaa aggttacccg 25200 ggccacatca atcttctttg ttggcttctg ggaagggat agctgtggtc tcgtcttgcg 25260 ctgcaagaca agtccatgtt agctcgctc tgggactcct tccacgccct cgacactcac 25320 ccctgctgga ggagactgtc acctgttctg atggggctt ccacttctgc cccttctgca 25380 ggaccagaa cacagtatct cttgccaggg cttggagaga ttcttctgtc tcaacaatcg 25440 tgccgtcttc ctccagcacg agggagaagg gcttgtcttt aagtttcaag atatcctggg 25500 cctggaaaag aaggttggtg acatttctgc catccctatg gagccacaga gttgaggagc 25560 aaaggcccgc agtaaagcac ttcagctgcc aagagagag caagtgagct gccatccatc 25620 cgtgcgatgg actgactgtc actgtgggg cccactcaag ggctgtgtct footcttcc 25680 tactcccaag aaggatgta gtctaataga atgggagtca tatgtgaggt ctttttttt 25740 gagaaataaa ttacatacca tgctgggcaa tattaggagt catatcgaag gccttgggca 25800 agctaggcaa actgcagcat tgaactatat ccccagcctt ctttgtactt tttgtttgga 25860 gacaggatct tactaagttg tttaggttgg cctggaactc actgtgtagt ataggtaggc 25920 ctcagccttc tatttagatc ttcttgactc agcctcctaa gtaacttgga ttacaggcct 25980 acactaccag gcccagctaa actttatttt gttggtggtg gttttttgtt tctttttgcta 26040 tgtagccgtg gctggccttg aactcacaga gatccacctg cctctgtctc ccaagtgctg 26100 gggttaaagg cacgttgtta tggaataatc ttttggtaca ctgtgaagtt gtgtctttgt 26160 caaggcgctt tctgactggt ttaataaaag aactgactgg ccagtagcta ggcaggaggt 26220 ataggcagga aagcaagaca cagaggactc tgtgaagaag ggcagagtct tgggagtcat 26280 gagcaaatgc agagggaagc aagatgaaag aaggtaccac tatgatgcag agggtagata 26340 gtaaaaggat taatttaagt catatgagct agctaaacac aactctaagc tatcagccaa 26400 gcatttataa ctaataatga gtctttgtgt agttattga gaactggcta tcgggataga 26460 aaagtctgtc tatagtgtgc atcaccatac ctggccacta ttttaaattt tttatttaa 26520 ttatgcgtat atgtatgtat gtgggtcccc tggagcaagt gattgtgagc tgcccagtgt 26580 gagagctggg aactgaacct ctgtccctg aaagtgcttt taacgactga gccaccactc 26640 tagccccaac aactcattg taatcttgtt cctgccaag ctacatggca tgaactgaag 26700 aaacaggtca ttcaaactg agggacagga aaaaata ctggcttgcc atctttaaaa 26760 gccagctaga agaatcagca ggtaagggta cttgcagtgc aaactttagc atttgagttc 26820 atacctgga gcccacagta gaaggagaga atgactccc aaatgttgcc ctgtgaccctc 26880 catgtgtacc ctgtgcatg ggcatgccag tgctcacaca cacgctcat agacttacag 26940 taataattt aaatataaaa taaaactgtc tggatttgca ggacaatcag cagtgctcca 27000 acagagtctc aagataagct tgggggtt cttgttgt acccctagtt gggcaggaac 27060 ccctatatag agaccaggct gacctcaac atgtgtcct cttgctctt catgcatcac 27120 cacactcagc cttagtttta atttcttttg aggcataccc agcgtatcct ggaactcaat 27180 tatgtagccc aggctagcct caaacctgag atcttccata tctggtctcc cagatacgag 27240 ccaccatgcc tggctcattt ctccacgtgt aaatatgggg aaggggaatg aatggccctt 27300 tcagcaagaa aataaccacc cacccacggt tggtgtgaaa gacggtagtg cactgccctc 27360 cttctgtcac ttgaccatgt cacttatcac acacacagct ctccaggcgg aaatgccaac 27420 cctccagtgc caacaccttt ctccgcttaa gaccatgcca ctcatgcgca cagacttcag 27480 ggctctccag aaaccgaagc agccggcagg ctcaactctc agccaaggcc tggagagagc 27540 ctatgcaggg taccggcaga tcccttacct tgcccaggag gtcctccagg ctgtgagcca 27600 tgatgccttt gcgaaccttc cgatctgccg tgctaactcg acagggcctc gccctgggga 27660 cttcccggct gggcttagac accagctgtt gggtcaccac tgcagtgctc actgctacat 27720 gcctggggac aacagtttgt acagcagatg acctgcctgc ggctaccagg tctgcccatg 27780 cctgcctgct tgctgagttc aggtgtgacc tttccttcca accaatcatg gctgcttcag 27840 gcggctttca caaagtccat cagaagacac agctccgatt tggaggggca gggaaatcag 27900 cgggaatctt gcttaaatca gactcacttc agtgcctctg agtggagccc aaggactgct 27960 aacaagtccc caagctgtgt cagtgtggct ggttcttgga ccagccacat tctcctgtgg 28020 atggcttctg cttttgtggc cttgttcacc ttgcctaaga ggcagggtga aatggagctg 28080 gtgtgtgtat atatgtgggg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 28140 tgtgtgtgtg tgtgtaaatc ggcctgtaga atatgtgggg actctgaggt acacagggta 28200 gaaagaaagg agcagagaga atttgggtct tggctggaat tccaagtttc tctctgttac 28260 ggatttgcta ggtgacagca tacccagcct gtgactcagt ttcctcatct gttgacttgg 28320 catagttgcc accacttccc agagaggagt gaggctcctg ggagatcagg aacaaaggca 28380 cctggtgccc agcctgaccc tttctactcc agatgtgatg ggaactgtgg cagtgacaga 28440 gagtaagatg gggactgtct agactgcaag atcttgggtg ctaccctagg cctcctgaat 28500 cagatctgtg ggggtttatt gtaagatttt gtctgttttg ttttaatggg ggtcttgctt 28560 atttgtttgt tttttgggat agcatctcca tatatagccc tggtttactg gaactaattt 28620 cctcatctct gtctctgtct ctgtgtctgt gtctgtctct ctctctctct ctggacctgg 28680 acctatgcag accagactca aactcaccaa gatcctcatg ccactgctcc ccaagagcca 28740 ggattcaaaa catatgccac catgcctggc catatcagct agaccccaca tgactttttt 28800 tgtcttttaa aatgggatgc ctatgtagcc ctgactgacc tgggacttgc tgtgcccact 28860 acaggctggc ctcaaactca gatctgcctg cgtctgcctc ctgagtgctg ggattgaaag 28920 cgtgtaccat cacatccagc ccctacatgg tttttatacc tattaaagtt ttcaaagttc 28980 taatagatgg ctccccagct ttccccttcc ttcccatacc cagctctcct tacctggaca 29040 gtgacttagg gtacaggagg ctgagagact tcatggcata gtccatcctt gtcagctgga 29100 ttgtgttgga cctggatcgg aggcagtaac ctatctggtt agagtgggaa gggagggagg 29160 ggtccctctc tttctttctt tgactcagtg gatcccagcc cccacacggc aaccctctgc 29220 cccagtctct cctcctaccc ccaatttccc tgaatccaca ccaactacac tgagtcagcg 29280 tctaccatcc ccacccattc cacagcccca gaacaactca ctccatgttt ctctgcccca 29340 agctctggtt acactgagct gaggaaccca agtcaaggct gaagaggccc tcactgattt 29400 gtccctgtcc ctgggggcag agtccacatt atgctggcag gaggtgagtc ataccacctc 29460 atgctgtgag agacttgaaa gtcacctatt gtccagaaac tccacaggca gtcagacctt 29520 ggagagcctg ctctaaccat gggcccttga gcaataaagt cctcctgagg cttagaactg 29580 gctaagagaa ctttctgtga tcttaaagac tgtctaggct tgtgttcacc aataccttag 29640 ctgttaccac tgagcccttg gcaccataac taatgtgaca aaggagctga caccgaactt 29700 ctattgaggc ttaactaatt tagttaggta actactgtgg tttgtgacgc caggtacccc 29760 actggacatt acagttgtag tggagtaatc ctgggctcat gtgccccctg cctttcctcc 29820 tatattgctg ttctctggtt cttgcctgct gagctgcaag tctctgcaca ggcagtctct 29880 tctacctgca atgcttgtct caaactcaga gctcaacata agtcacctag aagccccagc 29940 tcctggctgc caactcagga aagccttttc tggtgccctc ttgcctggta ccagattgat 30000 ccctgtgccc tgcccttcca tgtgaaagac ccctctccct tagccctttc tttctcaagt 30060 ttgtctcctc ttcctcctgt cggcggcttc cccgatcccc acccccggtg gcctttcccc 30120 gcccggcccg agaacaagca ccgggtgggg ccggcccgag aacaagcacc gggtggggcc 30180 ggcccgagaa tgagcaccag gtgggccagc ccgagaacga gcaccgggtg ggctggcacg 30240 agggcgagca ccaggtgggt cagcacgagg acaaacaccg agtgagccgg cctggagctc 30300 tgcccctgag cccccgcccc gcccgaagag aaacactccg tcccaaggtc tccgccccca 30360 aggtcagcca tcaggaaaag ggggggaatt gagtctgctg taccagacac cagaccttga 30420 gaatatgctg atctggaatg gctctgtgtc tcatttgaac catccaatgg aaatgattct 30480 gtatttcgcc tcatttgaaa gactctgtgt ttcacctcat ttgaataact ctgtactttg 30540 cctcatttga ataaccctgt atagcgcctc atatacattg accaatggga atagctctgt 30600 ataatgcctc attagaatta tccaatagaa tccttgctcc tagcttgcgc cttttttcct 30660 atataaggac cccttttccc ttggctcggg gcgcttagcc acacagaagc taagtcgccc 30720 caggtacctg cgtctccaat aaagcctctt gtttttacat ccagttcgtg gcctcgctga 30780 ttcctgggtg tgtgggtctc cctctacgaa agtgcctctt cggggtcttt catttggggg 30840 ctcgtccggg attgagaccc gcccagggac caccgaccca cgtctgggag gtaagtgttg 30900 tgcggatccg ctgttttgtc ttgtctggtc tgagtctgtc ttgtgaattg cgcttgcgtt 30960 tgtagtatac agctgtgtac atttgtaggc ggatccgagg agggactgac gggtccgaac 31020 tcccgaccgc ggctccagga gacgtcctgg tagcgtttga agccctcagg aagagggatt 31080 tgtattttga acttgggaag ccctcagggt gagagatttg tactttgaac ttagatctat 31140 gactggacat tttcccagtc tctttggaga aggccctcgg cttgagggat ttgcaatctt 31200 tactggggac gaggaaggag ggcccccttc ctcgactctc tctcaattcc ttctgtcgac 31260 tctctgtcga aaccgcgctg cgaaagtctg ttctgtgtta ttcggtcttt gtcttgtagc 31320 tgtcatttgt gccctcctaa gcctagaaac tatggggcaa actgtcacca ctcctttgtc 31380 cctaacactc tcccactgga aagatgtaca ggaatatgct cataaccaat ctgttaatgt 31440 gcgtaaacgc aaatggatta ctctttgttc ttcagaatgg ccgacctttg atgtaggctg 31500 gccgcgagat ggtaccttta acccccagac tatattccag ataaaagaga agattatgga 31560 tcctggacca cacgggcatc ccgatcaagt ggcttatatc gtcacttggg aggctttggt 31620 tcaggacccc cctccctggg tacgtccttt cttacatccc aagggcccct ctctccttcc 31680 cccctctaac cgctccaacc gacccattcc ttcggcccct acacctccca ctcctttgat 31740 tcctcccaac cccccttccc attccaacct ttaccctacc gtgatgaaag acactaaggc 31800 taaagaaaag aagacaccta aggtactccc tccgggagaa gaccagttgg ttgatctatt 31860 aacggaggag cccccgccat atccgccact gccgccccca ccagaggcag aagcggactc 31920 cgccgctgcc ttggcggaag cggcccctga cccttcacca atggcttatc gactaagagg 31980. tcgtagggag cagcccgttc cagattcaac cactctgccc ctccgaactg ggctgaacgg ccaacctcag tattggccat tctcagcatc ggacctctat aactggaaaa fatheratcc ttctttttct gcagaccccg tgaggctgac atctctcata gagtcggtac tcacgactca ccaacccacc tgggatgatt gtcagcagct tttgcaggtc cttttaacct cggaagaga acagcgcgtg ctactagaag cacgaaaaaa tgtcccagga gtaaacgggc agcccaccca gctacccaat gaaattgatg cggcttgccc tcttgaaaga cctgaatggg attttaccac cgaagcaggt aggacccacc tgcgtctcta tcgccagttg ctggtagcgg gactccgggg 32400 ggcaggacgc agacccacta atttggccca ggtgaagcag fathercagg gtgcggagga atcacctgcc gcttttctag aggregate agagcgtac aggregate ctccctataa 32520 tccggaagat ccaggtcagg ccaccaacgt ttctatgtcc tttatttggc aatcagcccc ggacataaga aacaagcttc aaaggctaga aaatctacag ggatatacac tccaagattt gttgaaggaa cgagaacgta tttttaataa gaggggaaaca cagacagaaa gagagaacg 32700 ttggaggaag gaaactcagg agagaga aagactaaga caggaaagctg aggaaaaaga 32760 ggttgcgaga gaccgtaagc ggaataaaga aatgagcagg ttattggcca cagatagtgac 32820 aggccagaga cagaatagac agagggatga cagaaggggg cccaccacctgg acagggacca 32880 atgtgcttac tgtaaagaaa aaagacattg ggcaagagaa tgccctaaga accccccgggc 32940 caagcttcca ccgccaaggg tttctgacct cctgaaccta gaagattaga ggagtcgggg 33000 ccaggagccc ccccctgagc ccagggataac actgcaagtc ggggggcatc cggtcacctt 33060 cctagtcgat acaggggcac aacattccgt tctgaatcgg tcaccccggac ccctgagtca 33120 caggactgca tgggtacagg gagctacagg cggaaagcag taccattgga ctacaaatcg 33180 gcagctccag ctcgcgaccg ataaggttat gcattctttc ctccatgtgc cagactgccc 33240 ctacccctta ctaggacggg acctattgac caaattaaaa gctcaaaatac actttgagag 33300 gtcagaagtc aaagtcacag ggccagaggg aattcccctt accatcttga caatgtccat 33360 agaatgaa tatagactcc atgaaagg gactattcg aacaatcagg aaacccttga 33420 tcactggctt gcggaatttc cccaagcctg ggctgaaca ggaggaatgg gccttgccat 33480 taaccaggcc ccaattatag taaccttaaa agctgccatc cttcctgcat ccgtcagaca 33540 gtatccaatg cctaaagaag cccgcgaagg aattcggcca catattaaaa ggttacttga 33600 acaagggatt ctggtgccct gtaaatctcc ttggaataca cccttgctac ccgttaggaa 33660 gccgggaact aatgactacc ggccagtaca ggacctgaga gaagtcaata aaggataga 33720 ggacatacac cctactgtcc ccaaccctta caatttgctg agtggattgc cacctaacta 33780 tacctgtac acagtcttag atcttaaga cgctttctc tgcctccgcc tgcatcccac 33840 cagccagcct atatttgcct ttgaatggca ggacgcggcc cttggaatct ctggggcagct 33900 gacttggact aggtacctc aagggtta gaacagccct accctttg atgaagcttt 33960 acatcaggac ctggcagaat tccgggttag gtaccccgct ctaatccctct tacaatgt 34020 agatgacatt ctcctggcag ccaaaaccaa agggaaatgc aaggaaggca ctcaagccct 34080 cctccagact cttgggagcc tagggtaccg ggcatccgcc aagaaggccc agatatgtca 34140 gaaacaggtg acctatttag gatacaagat aaaggatgga cgtcgatggc taacggaagc 34200 ccgtatgcga gccatcttag acattcccac cccacaaaat ccccgccaac tgagagaatt 34260 cttgggaacg gcaggcttct gccgcctatg gatccctggg tttgccgaaa tggcggctcc 34320 cctctacccc ctcactcggc caggggttgc ttttaaatgg gaagagcccc aaaagaaagc 34380 cttcaccgac atcaaaaagg ctctccttga atcaccagcc ctgggtctac cggacttagc 34440 taagccattt gaacttttta tagatgagaa ggagggctat gctaagggag tcctcaccca 34500 aaatctgggg ccttggagaa ggcccactgc atacctctcc aagaaattgg atcctgtggc 34560 atcgggatgg ccaccctgcc ttcgaatgat tgctgctata gccctgctgg taaaagattc 34620 tcacaagcta accttggggc agcctttgac catacatgcc cctcatgcag tagaggcagt 34680 catcagacag cctccagata gatggcttac taatgcccga atgactcatt accagactat 34740 gctgttagac aaagaccggg tccacttcgg gcctttggtg actctgaacc cagccaccct 34800 gctccccctc cctggggagc ccgaggctca tgattgctta caggtattgg ccgaggccca 34860 tggagcgaga tccgacctga ctgaccagcc tctacctagc ccggaccaca tctggttcac 34920 ggatggaagc agcttttttgc atcaaggaga agaaggcg ggcgcggcag tcaccacaga 34980 gaatcaggtc gtctgggccc aggcactccc ccctggaact tccgcacaga gggcagaact 35040 catagcactc acgcaggctc taaaattggc agaaggtaag aggctcaccg tgtatacaga 35100 cagtcgttat gcctttgcca ctgcccatat acatggagaa atttacagac ggagggggct 35160 gcttacctcc gaagggaaag acattaaaaa tagggagaa atcctcgctc tcttaagggc 35220 tcttcatctg cccgctgcct tagtatcat acattgccct ggacaccaaa aaggggattc 35280 tttcgaagca aggggcaatc gaagggcaga cttggctgcc cgagaggcgg ccctgaccac 35340 agacaccact aacctcctgg ctctagagcc caccaacgac catcccttcc cctcatggga 35400 ctatgaacaa agagacatcc aaaccctaga gaaattggga gccgcaaagg aaccaaacgg 35460 ggattggact tatgaggaa agactgtcat ccctaccgg gtaaccaagt acctagtgac 35520 atttttacat aagatgacac atctgagctc caagagatg cgggagctcc tcgaacgaga 35580 agaggaattc aatttccttt tggggagaa cgatattcta aacaggtaa ctgagcaatg 35640 tgatgcgtgc gcccgagtca acgcatccag actgaagctt cctcccggga accgggtcag 35700 aggctaccgg cccggacac attgggagat agatttcact gagatttaac caggaaata 35760 tggatacaag tatctattaa ttttgtaga cacctttca ggatggggttg aagccttccc 35820 tactaaacat gaacagcca agatcgttac taagaaattg cttgaagaaa tctttccccg 35880 ttatgggatg cctcaggtat tgggaacaga caatgggccc gccttcgtct cccaggtaag 35940 tcagtcagtg gccaccttat tggggattga tggaatta cattgtgctt atagacccca 36000 aagttcagga caggtagaaa ggatgaatag aacaatcaag gagactttaa caaattgtc 36060 gcttgcaact ggcactagag actgggtcct cctactcccc ctagcactct accgcgctcg 36120 taatacccct ggaccacatg ggctcacacc ctttgagatc ctgtatggag tacctactcc 36180 tatcattaac tttcttgatc aagatgtctc agattttgct aactcccctt ctctccaagc 36240 tcatttacag gccctccaac tagtacaacg ggaggtctgg aaaccccttg ctcaagctta 36300 taaagaccag agggaccatc ccaccatccc ccattcctac cagatcgggg acactgtttg 36360 ggtccggcgt caccaggcca agaaccttga accccgctgg aagggaccct acatcgtttt 36420 gcttaccact cccaccgcac tcaaggtaga cggcattgca gcttggatac atgcttcaca 36480 tgtaaagcca gcccaaccca ccgattcagc cactgcatca gaatggaccg cacaccgcac 36540 tcaaaatcct ttaaagataa gactctctcg tacaccctcc tgttgattgg ttgtctgttt 36600 accccccatg tagcaactaa cccccacagg gtttataata tcacctggaa aatagccaat 36660 ctagggaccg gggaaatagc caacctcagc acttatatag ggactctaca tgatgggttc 36720 cctcctctct atgtcgacct atgtgactta gtagggtctg attgggatcc ctctgaccag 36780 gaaccattcc cagggtacgg atgccaccac cctgggggaa ggataggaac aagaagcaag 36840 gatttttatg tttgccccgg cataaacca actcatggct gcggggggcc gcaggaaggg 36900 tactgtgcaa gatggggatg tgaaaccaca ggggaggctt actggaaacc ctcttcctct 36960 tgggatttca tcactctcaa acggagggag atcccagggt acgcaggggaa aggaccatgg 37020 agatgtgggc aaagagcctg cggaccttgt tatgatagtg ccggaggggg aggttttcaa 37080 ggcgccaccc ccggaggaaa atgcaaccct ccatcctaa ggttcacaga tgctggaaaa 37140 agaactactt gggatagtcc taaggtctgg ggactcaggc tgcaccgagc agggaaagat 37200 ccggtgactt tattctccct gtacagacaa attactcccc taagccaaca atcagttggg 37260 ccaaacatag tatagcgga ccagagatcc ccaacccatt ttcaagtccc taaaccccct 37320 accgttccta aagctatcac tcctacacca ggtgctgtca ccttctcccc caccccagat 37380 gccctaaaca tcgagataac cagagaccct ccaggtacca gagatagatt attacaatta 37440 atccaaggag tttaccaagc cttaaatttt tcagacccca acagactca ggaatgctgg 37500 ttatgcctag ttcccggcc cccatattat gaaggcgtgg caatactggg caactactcc 37560 aaccagacct cagcacctac cagttgcgga gctgctatgc agcacaagct cacaatctct 37620 gaggtctcag gaaaggggct atgcataggc aggattcctt cctcacatca agaattatgt 37680 aaccaagtag agccattatc tcaggacagc cgataccttg ttgcccctta tggaacttat 37740 tgggcttgca gtactgggtt gactccctgt gtctctacca ctgttctcaa caccaccatt 37800 gacttttgta tattgataga actttggccc aaagtcacat accaccacc tgaatatgtt 37860 tacagcgtac tagagaatc aacccgatat aagagggagc caatacctt taccgtggcc 37920 ctattattag gaggaatac atggggggc atagcagccg gcatagggac cggaaccgtt 37980 gccctacagg gatttaatca ttttaagctt ctacacaag ccatgcacac ggatatccag 38040 gtcctagaag agtcagtcag tgcactcgag aaatccttaa catcactctc tgaggtggtc 38100 ctgcaaaaca gacggggatt agatttatta ttttacagg aaggggggct atgtgctgcc 38160 ctcaaggaag aatgctgctt ttatgcagat catacagga tagtaggga tagcatggcc aaacttaggg agaggctaaa acagaggcaa cagctatttg agtctcaaca aggatggttc gaggatggt tcgctaartc cccctggttg actaccctta tatccacgct catgggacct 38340. ctggttattc tatttttgat cctcatattt ggtccctgca ttctgaacaa actgactcaa ttcatcagag aacgactatc tgttgtacag gctttagtct taactcaaca atatcatcag ctaaagcaaa tagatccaga gtatctagag acctctgaat gaaagattcc attcagttac aagagaaatg ggggaatgaa agacccctct cccttagccc tttctttctc aagtttgtct 38580. cctcttcctc ctgtcggcgg cttccccgat cccccccc ggtggcctttt ccccgcccgg 38640 cccgagaca agcaccgggt ggggccggcc cgagacaag caccggggtgg ggccggcccg agaatgagca ccaggtgggc cagcccgaga acgagcaccg ggtgggctgg cacgagggcg agcaccaggt gggtcagcac gaggacaaac accgagtgag ccggcctgga gctctgcccc tgagcccccg ccccgcccga agaaacac tccgtccccaa ggtctccgcc cccaaggtca 38880 gccatcagga aaaggggggg aattgagtct gctgtaccag acaccagacc ttgagaatat 38940 gctgatctgg aatggctctg tgtctcattt gaaccatcca atggaaatga ttctgtattt 39000 cgcctcattt gaagaactct gtgtttcacc tcatttgaat aactctgtac tttgcctcat 39060 39120 cctcattaga attatccaat agaatccttg ctcctagctt gcgccttttt tcctataataa 39180 ggaccccttt tcccttggct cggggcgctt agccacacag aagctaagtc gccccaggta 39240 cctgcgtctc aataaagcc tcttgttttt acatccagtt cgtggcctcg ctgattcctg 39300 ggtgtgtggg tctccctcta cgaaagtgcc tcttcggggt ctttcacatg ttcatttctg 39360 ctgcagctct cgggtcccca ctgtgcctgt tttcagcccg tagcacaggg acccagtgtg 39420 aacaccagga gtgggcaggc atggagaact gcctcctcag tgaaggaaaa ccattcttcc 39480 cttctgataa ctcgggttct ccccggtcaa ccttacagag ttagagaccc tcgtccccac 39540 ttaatgctac ccaagcactc ttgtttcacc ctgtttctaa tacccaccac tcacacagct 39600 ccacaaaacc caccacacct gctctgtggt agccaaatac cccagctctt ctcctcccct 39660 cacctcctag cctcctggca atactgcgtg gccaggcaca gcccaggcct cctccatttt 39720 atttctatct cctgtgaagc cactcccttg gccccagccc cagctggggga aaagagcaca 39780 ggagtgagtc tgaaactctc cctagggatt tcaagccaga ctgacttcca gaaagccccg 39840 aaagggaag aaggcatgtt tcgaaagctc tagaaggaag aggtactgcc caggacttgg 39900 taccaggcaa gagggcacag aaagcaacag acggggcttg tggtctgcgt tccacccctc 39960 acaccagctg tttgcgctca cccgagttgc cttctttcag ccctggattt tctcctgtgc 40020 atgaggaata acgctcctgt tgaagccagg aatggtggct cagagctgta atctcagcac 40080 ttggtagact gaggcataaa aagggccatg agtttgaagc tagcctgggc tatagagtta 40140 ggtcctgtct caaaaacaaa aatatagtca gacggtggtg gcacacactt ttgatcccag 40200 cactcggaag gcagaggcag gcggatctct gtgagttcaa ggccagcctg gtctccagag 40260 cgagtgccag gataggctcc aaagctacac agagaaaccc tgtctcgaaa aaacaaaata 40320 aataaataaa taaaaaagaa atatcttttt ttttttgagt caggcaagat acttgagttt 40380 tattatgtct gtctaaaagc aggtgttagg cctatttgta gtgaagcgtg gtgtgctggc 40440 gctgcaacac cggtgaggaa gcaggaacct gatctcagag cccaagaact gcttgacagc 40500 aggccttcgg cacttgccct ctgaaatctc ttccaccttc atcaggttaa tggagtgtgc 40560 acgggcacag tgcgggcacc cgtgtcttgg tagcactgtt tgacggctcc agcagcggtc 40620 aggtcccggt attcttggca catgttgtgt gtgtcatgtt gtgagttgta gtgcagccat 40680 accgcaaagt tcttcacccg cagtggggcc tcaggtcgca ctgcacagtg tacgatctcc 40740 tcggatgact tcttcatctt gttcatctgt gcccaaaatc gggacttggc caccacatgg 40800 ttcgtcgcaa agatgtgcat gaggtatagg agtggtgtgt ggcacttccg ggtgggcaag 40860 cagcgcccca ccaccttgta ctcccgaagc gtgcctgagg ccttcgtggc tgatctgtcc 40920 ttgttggccg ccagccacaa aaataaaagg aaatatcttg cctataatgt tacaatccca 40980 tccagcactt gggaagccaa ggcaggaagg cctctgtgga tgagaccagc ctgggctaca 41040 gagtgacaac ctgccaagag aatggggtgg gtaatcccaa atcttgggta tagtgaagta 41100 caggactcaa gagacagagg caggaggatc atctccagtt caagacaagc ccgagctata 41160 aggcctcaaa aacaaaaaaa gaatattaga tctatgattt attgctataa acactacaac 41220 ttaaaaaaaa ttaaaataag gacagcaaga tgactcagtg aatgagggtt tgaatttgat 41280 ccccagtacc cacatagtac aaagagagaa ctgactccca taggttgtct ttccgtgaaa 41340 actctccctc ccttcctccc tctctctttc tctctctcta aatgaaaatg tttacatcaa 41400 agcctctaca aagagcattc atgttacacc tagcgttgga gagatgtctc agtggtcaag 41460 agcgctgact tctcttccag aggatccagg ttctattaca agtacccatg tggcaattca 41520 caatcattta taactcagtt ccaggggatc tgactccctc ttcaggcctc tgtgggcctt 41580 catgtgtgtg gtgcaagac ttgcacgaag gcaaacact gtacacataa aaaaaata 41640 agagacagaa aagggaattt attcagtgtg gccataccag aaagatgag agggaccagtc 41700 tcttcaaccc tgtttttgga gtgcagatgg tagctctagg aatttacagg agagaacaa 41760 aaaatgggat gagctggtgg tgcaggcctc tcagtgcttg agaggcagag gcagaggcag 41820 gggaatctct gtgggtacaa ggacagcctg actgacctgg tggattccgg gctaccgaa 41880 actacaaat aagacactac ctctaaagtt aaaaaaaaa agggaggcaa taagtatgca 41940 tagctaagca ttctattcta ttctcccaa accactcta tgacggcta cctccagtct 42000 gaagtaaggg tcctgcagta gctaggaaga ggactacctc ccaggctgac tgttcctggc 42060 tctggcaca aactgaaag gaaaactggt tcagaggag atcagagcag aggggccaaa 42120 ttagaacgag gattgtgcct tccttcaggt ttagacac cactggagaa ttttagtac 42180 tgctgtgta gtttggaaca agacgaggca aaacttatt actggcaatc tgtaatgtcc 42240 ccataaatcc ttttatatc tattttaaa aagatttcta tattatatgt acagcattct 42300 gtctgcatat atgcctgcag gccagaagag agctccagat tttattacag atcgttgtga 42360 gccaccatat ggttgctggg attaaactc aggacctctg gaagagcagc cagtgctctt 42420 aacctctgag ccatctctcc agccctctat tttatttttt aaaagaatta tttatgtgt 42480 atgaatattt gcatgtatgt atgtgcacca cattcatgca gtgcctatgg aagccagaag 42540 aggacgacag attccctgaa accagagtta caggggctgt gagttgcttt atggtgctga 42600 gaactgagct tccttcctgt acaagaaaag cacatgctgt taaccactga gctgtccctc 42660 cagccctccc cacccctttg tgtgtgtg tacttgtatg tgtatgtatg tggacatgtg 42720 agcatgtgtg gaagtcagag gatggttgc aggagttggt tctctccttc taccctgtgg 42780 gtcctaggga taaaactcag gctggtagca tgtccctcta cctgcaaagc catctcctgg 42840 gctttgtccc aataaatttc ttgctctccc ttggtatctt cattcttaaa gggaaggaca 42900 gagctacagg tacaatgggt gaaaattggc ctttagttac cagtcttgaa gatgagtact 42960 taaatttttt tcagaatcct tttttttttt ttaaatcttt ttatttatgt atacagtatt 43020 ttgtctgcat gtatgcctcc aggccagaag agggcaccag atctcataat ggatggttgt 43080 gagccaccat gtggttgttg ggaattgaac tcaggacctc tggaagagca gacagtgctc 43140 ttaacctctg agccatctct ccagctcaag aatccttttt tttttttttt ttttttaagg 43200 cagggtttct ttgtgtagcc ttagttgttc tggaattagg tttgtagacc aggctggcct 43260 gaattcaaag acccacctga ctctgcctcc cagggtgctg ggactaaagg tgtgtgccac 43320 gacacctggc taagatgagt gctttttaaa tgcctatcac atctacaggc acaactgatt 43380 gggaagctga ttcaaacctt gagatagcaa agggatagag gtgttgatgc aacataaaga 43440 cagagatgcc cagattttct ccttccttta gtcactgggc gggactctgc aggtccagtg 43500 aggactcact ggctctcttg ttaaagcctt atttagagtt aagccaatcc ttggaagagg 43560 catcattcat tcattgaaca cttgcagtgt gttaccctgt taattcagca gaaagcaaga 43620 cagctttagg ccacacctgg agcagctgga ccagagacca agtggaagca ggaatgctga 43680 tgtacacttc tcatcctgat cccctggagg ctgtcagcct gggccacaca gtgcgagatc 43740 ctgcctcaaa aacgtttttg ctacccattg gtgattgtac atgtactttg ttctagcact 43800 caggaggtag ggccagcctg gtctacaaag gacagccaat gctacacaga gaaaccctgt 43860 ctagaaaaac aaaaacaa cagaaacttt tttgtctgat gtttcatctt gttctttttc 43920 tataatgta ctgggcagag aaaaacaaaa taatgcagtt gggcctggag gcttagacct 43980 gggattccag cacttaagag atagaggcaa gctcattgct ttgagtttaa ggtccctctg 44040 ccctgcatag tgagttgcag gtcagtgtgg actatagagt aagaccttga ctttcaaaaa 44100 gcacaagggg ccaggcgttg gtggctcaca gctttaatcc cagcactcgg gaggcagagg 44160 caggaggatc tctgtgagtt tgaggccagc ctggtctaca gagggagttc caggacagcc 44220 tccaaaacaa tacagagaaa ccctgtctca aaaacaaaac aaaacaaaac aaaacaaaaa 44280 tgtcaaaaag cacaagggcc atcaaaatgg ctcagtgggc aattacaaac ttgatgacct 44340 gagtccattc cttgagacct acataatgga aagaactgac tcccctaagt tgtcttctga 44400 ccttcacaca taccgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 44460 gtacatgcac gcgctctcgt gagcaagtac gctcctgata ctaaattaat aaaaaggatt 44520 aaaaaagtaa aaccacaaac atgaaccaaa ccaatgtggt gtaataacct atgtctggag 44580 aactaacagg tttctggacc cagagaggta gagaagataa tctaatgtct tcaggggaat 44640 tagggggata aagcatcaga gcataaagaa agttccctc acttgggaag tagtggaggc 44700 aggagcatca ggaattcaag gtcatctcat ctatatagta agtaagttca agactagcct 44760 gggttacatg agaccctgtc tcaaaaaaaa aaaaaaaaa aaaaaaaaa aaaaaaaaaa 44820 ggaagcatgg tgcaacagat ttgagattga aacacaaaa tagctcaact ctggggcaga 44880 cagcagagtc aggggaaagt ctaaggaagt tctggggaaa gggtcaggag gcccctccct 44940 gcttcattca cgccccaaac atctattaaa cattccatgt gtacttggtg tttctggcct 45000 gaggatgact cacagaggct gacacactaa gttaatcaca ggaagcacag aagaaaaaaa 45060 aaaaaaaaag aaaaaacagg cctaatgcaa tggaatgcct gactgagggg gttcttcaca 45120 gtgctttgga gacgaccctt tgtgtctcat tgaaattcat ctctcaagaa acactcctca 45180 aatgtttaca cattgtcagg cactttgagc tgcagggaca cagaggactc agttctggtc 45240 cctgcctttg gaattcacaa ttgttgctct tccaaagaac tggggttcgg ctctcagcac 45300 ccacttaagg aggcccatga ccatttgtaa cttcagctca accgcttcca caagtaccca 45360 cacacatgtc tgtcccccaa acacacctat attcttcatg ctaaccttgt gacactatga 45420 gcaaaatcaa agacattaag aagccaggtt cagtgccctg tgccttagtc ctaacatttg 45480 agaggctaaa tcaaacagga attgaggcca cttggacagc atcaagagaa attatctttg 45540 aacatatgaa atgatattat cacagaaata gaattgcaat ttaaaccagg tatgtgatcc 45600 cctctccccc caccaaaaga aaaaaaaata gagggctgga gaaatggctc agtggtaaag 45660 agcacctgct gttcttgcaa aggatctaga ttgttttgtt ttgttgagac agagccacac 45720 tatgtaactc tggctgtcct ggaattcact atgtagatca ggctagctgg tatgtatgtc 45780 tgtccacaaa tgcatgcctg gtgttagagg cttccagaag tgtgtgtcag accctctgga 45840 actggagtta taggtggttg tgagctgcca tgtgggtgat gtaattgtac ttgggccctt 45900 tgcaagagca accagtgcccc ttaacccctg atacttgctt ttaaggaatt tgcatttttg 45960 ttttgaaatt acatgttgga aaagttttcc acatcagtaa gaaatgccat ccttactatt 46020 tcttcctgca gcttctcaga aatatttttt aatcttttttt tttaagattt tatttattta 46080 tttattatgt acacaacatt ctgcttcatg tatatctgca caccagaaga gggcaccaga 46140 tctcattcaa ggtggttgtg agccactatg tggttgctgg gaattgaact caggacctct 46200 agaagagtag tcagtgctct taacctctga gctatctctc cagcccctca gaaatatttt 46260 aattcaaaaa tctttcttag ccagctctca agccactgga ttacttcttg cttctgctac 46320 tgacatagac ttcatcttga ttcactccat acagcaacta gatgtgtgta tataaatgac 46380 agatagatca tagatggatg gatggatgga tagatagata gatagatgat agatagatag 46440 ataatctcac ttgctaccta ggctgaccctc aaactcatgc ctcagttttcc taagtgatgt 46500 gattacggggc atacactatt atgcctagca taaccattg ttgccttaa aattttaa 46560 gatttatttt ttattatgta tacagtattg tgcctgcagg ccagaagagg gatcagatc 46620 tcattacaga tggttgtcag ccaccatgtg gttgctggaa cttgaactca ggacctgg 46680 aagaacagcc agtgcttta gccctgagc catctcca gccccagcc tttaaaattt 46740 ttaattaatt ttttttgt tgtgtatgg gtgttttggcc tgcatgtagg cctatgcact 46800 atatgtgtgt agtactcacc tagaccagaa gagggtgcca gagactctgg aattggatt 46860 tcagtgcatg atgagctgcc aagtggggcct taatccccaa accccagagg gttgaagagg 46920 gatcagtgc taagggtgct tgttcttgca gaggacccag tttgattcc cagagcccac 46980 atggtagctc agaacaagaa ctccggcttc agaggattct gcaccctc tggcctcat 47040 ctggtaccag gcatacatct ggtatgcaga tatatacacg ccctgagtgg tgacaacac 47100 tctgtgagtt caaggctggt ctacatagag aattccagga catagtgaga ccctgtctca 47160 accaaaac ctggatcctt tgcaagaaga gcaggtgctt ttaaacactg ggccatctct 47220 ccagcaccac ctcccccctt taatgataca taggtcccac atagcctagt tcggcctctt 47280 aactcctgac ataacacctc atgaattcct catcctcctg cctctacctt ttgagtgagt 47340 gacgagatta caaacgttcg ccaccatctt tgttcccagt tggccctcaa ggccccaggg 47400 tctcagttca gatcctgcca ctatggatac atagcattga aatgggggaag gctgcctctg 47460 tgctgcctca gcctttgggt gacgcctctt gcttggcact gcattttcag gagcatgaca 47520 tctttgctct ggaccctgac cccaaggttg tgtggagctg taagaagtgt aagggtgtct 47580 ggtattggtg gcgcgcgcct ttaatcccag cactcgggag gcagaggcag cagaggatct 47640 ctgtgagttc aaggccagac tagactacag agcgagttca ggacagccag ggctgttaac 47700 acagagaaac actgtcttga aaagcaaaac aaaaataaaac caacaaaaaca acaacaacaa 47760 caaagtgtta agcgcgcatg caaccagcca ctgggcaatt agtcaaagat gccaagtcta 47820 gatagacatg cagttaagaa cttagggtct agagagatgg ctcggtggtt aagagaccag 47880 aagagtttgc atcccagcgc tcgcacagta gctaacaaca gtctatctta caccctttc 47940 cagcctctcc tggcacaagg aacacacgtg gtgctcatag ttacaaaaca gacacaacac 48000 gcatacacaa agaaataact aatttttaaa atgtcttgta atcaagtaaa agtgctaact 48060 ctagggacta aattaattcc tcagcgccc tactccagga gcggtaaggc tggccagaaa 48120 gaccaagaac gcacgcgcgg aggagaaacc acagagtcgg tcctcccggg atagagaggc 48180 cggaagtgct cgcggagctg cacgcgggt gctggaagcc tactgagccc cgaggaaggg 48240 ctccgctcgg ggcttggcgt ggtgggtgag ccggagggtc ggcgtgagcg gcctgggctt 48300 tggttctgaa tgatggcgtc tcgggcaggc ccgcgagcgg ccggcaccga cggcagcgac 48360 tttcagcacc gggagcgcgt cgccatgcac taccagatga ggtatgaggt gagccaggag 48420 cactgaggcc ttccccgggga ggagcctgcg ggtctcgggga agcgacgcgg gcgagcctca 48480 cggtgccgct cccccagcca gctgtcgcgt actaccgggt ccccggctcc ggcgagcgcc 48540 tcgggtctgt ttacaggccg ggaagcccag tggcctgccc tcgcccgcct cgtgctttga 48600 ggggatctgg cctgcagagg ctcaggggtc gatgctcagc ccctctgaat gaccttggag 48660 acatcatttt tcttttttca aatcgaggtc ccgcagtgtc tagagttcag gctggcctgg 48720 aactcacggc cttccctcct cagcctcccg agtatgcgcc tggtctgaaa actaacattc 48780 ttaaagaaaa ctgcgtgtgt gcgtgtgtgt gtgtgtgtgt gtgtgtgtgt gcgcgcgcgc 48840 gcgcgcgtgt gtgtgtttgt gtgtgtgtaa gtttgcccac acgagagcag gtgtgctggc 48900 agaatgcagt gccagtaaag ggtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 48960 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg cgcgcgcgcg tgtgtgtgtt 49020 tgtgtgtaag tttgcccaca ttcttaaaga aaactgcgtg tgtatgtgtg tgtgtgtgtg 49080 tgtgtgtgtg tgtgtgtgtg tgtttgtgtg tgtgtaagtt tgcccacacg agagcaggtg 49140 tgctggcaga atgcagtgcc agtaaagggt gttggatctc ttggagctat agttacagac 49200 gtgtttaaat ccacctgaga ctagtactga ggaccaaacc ccgaagtgat tttgtgagat 49260 agcggcttgg tatggggtcc atgctagcct ccaggagcgg caggtgctct taaccactga 49320 gccatctctc cagtccccag gctgtcttgg aacttgctct gtagaccaga ctagaaatca 49380 gagatctgcc tgcctctgct gccaagtgca aggattaaag gtgtacacta cagccgccct 49440 gttgaccctg tctttaggaa gatgtcatgt gtactttaca aagagttttg atgtagtcat 49500 gtcttaagct ttgttgtcac acaagatgaa attaattacc accagggaat ttttcatcaa 49560 attgtgctgt acttaaatat gcctaaagta aaatacttgg ggtcagactc ttgaagttta 49620 aaccaacacc aggtactgag ttgtgttgaa cccacacaac taccttgcct cctgcagatt 49680 gttaactctg ctggctgtgg tggttgaaga gacccccaca ttggcttagc tgttaagagc 49740 acttgctgtt cttggagagg gtctaaactc agttcccagc accgacatca ggaggctcac 49800 aaccaaacac ctgcaactcc ggttccaggg gttccgatgc cctctggctt cccgacagca 49860 caggcgtgtg cgtggtgcac acacatacac atgagataaa tttttaaaaa gaaagaaaag 49920 ccaatctggg tcctgccttc aaggagcttg agtgccagaa gtgattcttt tctccttacc 49980 tcccacattt cctataacaa attaattttc cccttaaatc cactgaatta ttcaactggt 50040 tcttggcctc tttctttaca gaaggatgac aagccctttg gacccttctt tttcaacttt 50100 aggcccctgc ctaatggatg ctcttgtttt catgcgtacc tagagcagac accttgcttg 50160 tgacaatatg aaaaggatat aattgagacc ccgagaagat caggactaag agatgtgcct 50220 ccttctcatg gaaactatat ctggagaaat acagggagcc caaactgtgt tccttgttgc 50280 taggcctccc cctaatggcc tttgtttctg tgatacgaag ccatctttct aaccacagct 50340 cagaattctt gtcatggctt ggtgagataa tgtcttagta cgttgtccag gccaacctcc 50400 aatcagagat cctctgcctc agcctcccag gtgccattac tgttcttcag tcttgaagtc 50460 tacacatgca tggtctttgt aactgttact acctctctgt gccttggcag ttgatttcct 50520 gaaacacaaa tactaaaaag cttattcctt tgtgcctgca tgtttttgcc tctgcgtaga 50580 agcccacttc cactcgtctc attacctctg ctgaatagc tctctcctct agatataacc 50640 ttctctggct ttccatctca tgctaattg ttcttccctg ggatagcgga tatcttcggt 50700 ttatatgtat ttacttgagt tttatacttc atgtttccca gcctctatca gaggctgtgt 50760 cattcagatt gtatctatgt gtccagtacc ggtgcctggt ggtcggagca atttaaatga 50820 aaattgcttc tccctctgct ctgccttaca gtgtgacgct caagagtgaa atcaagaagc 50880 tgatctacgt acatctggtc atatggctgc tgttggttgc caagatgtgt gtgggacacc 50940 tgaggctctt gtcacatgac caagtggcta tgccctatca gtggaatatc catatttatt 51000 gagcattgtg ccctctct tgggccttct ctccttcctc gaaacaacat tagctacctg 51060 gtgctctcca tgatcagcat ggggctcttc tccatcgctc ccctcattta tggcagcatg 51120 gagatgttcc ctgccgccca gcaactctac cgccatggca aggcctaccg cttcctgttt 51180 ggttctccg ctgtctccgt catgtaccta gtgttgatac tggcagtcca agttcatgcc 51240 tggcaactgt actacagtaa gaaactctta gactcttggt tcaccagcac acaggagaag 51300 aaacgtaaat gaagcctgcc tgatggacac atgaagggag ctgttcagaa tctccatgga 51360 ctgtggcatc tgtgatgttg gcacctagtg cacactatcc tcagattttg gccttgagtt 51420 ctctgttacc atctgctgag atgacaaatc tgtagtgttt aatttattct tgactagcca 51480 caaaccggat gaccgatgtc tgtggaacac ttcaagttga ggccctccag actgagcctt 51540 atccttgcct tctcttggtc aaattcctta cttccattta tcacctcttc atcaccgata 51600 ctaaaagaga tctggaataa atcagtgcag aaattctact tcaatctgta ggtcatgggg 51660 caagcaacat ttgggaagtt gctcccctaa aggctactct gtttactgca aaccatttta 51720 attaaaaaag aacttcaata aagttaagac tgtcctgtgc tttgtgtttg agatttgatt 51780 gaatttcaaa gttgtattgc tctagaggac ttagacatta tgagaagaat agatgtttcc 51840 tgagaacatg ggactggcgc tggaggagag acagtgtgaa gagtcttaag tcagctgcag 51900 ccctctagct cttaggtgag aacaaccctg ggatgggggt tgctggagga atggctcagg 51960 ggttaagagc actgacagag gacacaggtt tagttctcag cacccaaatg gtgacttaaa 52020 accatttgta actactgtcc cagggcattt gacgccttct tgcctccgga tgcactaggc 52080 agacatgagg tacaaaaaca aacatgcaag caaaacactc aaaaaattaa aaaaaaaaa 52140 catggtctct caatgtagct gtggatgtac tagaactcac taagtagagc aatctgggct 52200 caaactcaca gagatcctcc tgcctttgcc taccaagtac agggatttaa ggtatgtgcc 52260 accacactta gcaatatata tttggccaga catttaatct ccacccataa agcagcttat 52320 ttggcttgga ttgagctc ccctttttat gcttgtttga gacagggctt cctatagct 52380 ctggctgtcc tggaacttgc ttcgtggacc agactggcat caaactcaaa tccacctgcc 52440 tctgcctccc acgtgctggc attaaaggtg tgtgcatcac tgcccagctg ggttacgagc 52500 tttctatttg gttatttatg tttattagac ttttatgtgt ctaaaactga gtaataatac 52560 ccaaaaagaa tctgcttgtt ggggctataa ttttccagta actgacaaat aaacctgagt 52620 aaaaaagagggt ttgagttctt tttccctttt agtttctgtc attgatttt 52680 ccttggcctg cttgtttggg gaatgtgcaa aggaagctgc taactctatg gaagccagaa 52740 aaagaaaaag gtgagattcc agtgtcgcca ctaggggcag actgccagtg acgtaacttc 52800 ttcactcagc ccctcccaaa gcttcctcca cctccaaaaa tgtcaaggac tggtaactag 52860 tacatgggcc tttgagaaaa gatagcaggg agcttccagt cttgggggaa tgtgcagtga 52920 52980 gaagtcttca catataa ggcagcaaag gtgtgcatcc ctttgcactg gactttatta 53040 tgcctagatg atgctcagat ctgtaactgc agagatctgg ccactacagt tgtaattctt 53100 ttgtcctgga aatgtgttgc ttgtacagtc tgcaaagcta attcctccag ctatcccact 53160 gatcatttct ttggagcagg ggcagtttcc agacagaatt tctctgtgta gtcctcgcta 53220 tcctggaact cagagatctg cctgctgtgt gctgggacta aaggcatgta ccaccatgcc 53280 cagctcctac tgatcatttc aaagataact tcttgtgcct caggcctatt gctgttcctg 53340 cttcctgtgt ttacgggaac acagctctgg atcatccacc ctgcaagttt aagtccgtcc 53400 ccactccata tacacactgc ctattcggtg aacctcctat agataccctg ataagttccc 53460 tagcttctca gtttaacact tagaactctc cctacttaag ggaggcctct cagtggtgaa 53520 gtgctcctct agcatgcacc gaggcctggg tgtgactccc tcgtaacaac aaataaaaac 53580 aaaccttttc tgctcttaca ttcctttatt aatctttaag atgccacttg tatgaaaatt 53640 cctcaatttt caattcctca atttgaagaa caactttaat ttgtctggtt gctttcacct 53700 tattccttta ttattatttt ctaacaagac agagcccaga atgaccttaa gctatgtggc 53760 tgaggatgac tttgaactcc tgatgctttg tctctacttc ctaagtgcta ggattacaag 53820 tatgcaccat cactcctggc ttatattact cgtcccccac ccccacccca tagaccaagc 53880 cttgaccttt atttttaaaa gctgcatatt tattctgctt tctgacttag tgtttgtgcc 53940 ttcaacactt tcacaaccct tttctctcct caataaggaa agcctgcttg atcctgtcac 54000 ggacacactt ggcacacaag gagccaccac agcccctgct gacgtgcttc tttgtctaga 54060 cagtctcata aggactttgg gtctcacagc atgaaccctt caagtctgcc tgggcacaca 54120 ccacatgggg attcaggtgc tttcccagtc ttcttggtat aaaggtaaac aatcctgttg 54180 ccagggttcg agacagccta gtttcgttag aggctgtatt gtaggaaagc ctacaatggt 54240 atgtcaaaca ctggaccatt cgagtgcctc taagcaccat ccctggaaga ggaagagctg 54300 tattatttgt tttatattga cagaattgct actttgtatg tcttctgtag ctcattagtg 54360 ttcactctga gcttattgaa tgaattaact tgccctttg aggacaaagg tctgtatttc 54420 acagaaaaca gaactggact gaagcagaaa gaagcccatg gacaagggag tttgtcatga 54480 gcttagctga ggcacacatt gctaacttca aaaactgtg atgcaaacaa ttaaaactgc 54540 agtgcagacc cccctgcttt ctcccaataa atttccaca tagttctgt ccccccccca 54600 aaaaaaaac ctgttagcac attattagtc aaagagctaa aacctgtttt tggtcatcag 54660 aatagtgttt tactgaaagt ctttgaatac cttgtagact aatttcactc attgtgaaat 54720 tagtcaacag ttttaaaaca ctatcatgcc aacatcagtt tttgttagtt ttgtgcttgg 54780 aattctgctt ttctagcctt ttcctataac ttttttctgc ctcatggtta tggtgttcac 54840 aagtcctctt gatttagtgg gaagaatgct atttgggtta gaaagttcag ctgggtatgg 54900 gggtacatgt ctaatcccaa cctttggcag gttgaggcag caggaatact ctgagtttga 54960 ggccagcctg acatacatac atacatacat acatacatac atacatacat acatacaaac 55020 actatctcaa aaaacaaagt tggagttaaa gtatcaacca caggactaag atcaacctgc 55080 ctgttcacag gttgctttaa agctccaata gccggttgga gagatggctc agaggttaag 55140 agcactggct gctcttccag aggtcctgag ttcaattccc agcaaccaca tagtggctca 55200 cagccatcca tttgaggtc aggtgccctc ttctggtgtg cagatgtaca tggaagcaga 55260 atgttgttac ataataaata cataaaatct taaaaaaaaa aaactccaat agccaaagta 55320 tttgaaggc attctaaaat ttttaattta ttttcatttt atttttggag acacagtttc 55380 tctgtgtaaa aaccccaact gtcctggaac tcactctaga ccagactggc ctccaactca 55440 cagagataaa cctacctctg cctcccaagt actgggatta ggccgggcag tggtatcgcc 55500 cacctttaat cccaacactc gagaggcaga ggttggtgga tctctgtgag tttgagacca 55560 gcctggtctg caagagctag ttctaggaca gcctccaaag ccacagagaa accctgtctt 55620 gaaaaaaaca aaaaacaaac aaaaagcaag ttaatctgga attaaaagtg tatgccacca 55680 cacctgatgt aaatttaaaa ttttttttaa attaattttt ctatacacag gtgttttgcc 55740 agcatgtatg tttgtatagc acatgtgaac ctggtgccta aaaagccaaa agagtgcatc 55800 tgatcccctg ggactggagt tacaaatgga gtgctgccat gtgggtgctg agaattaaaa 55860 ccaaatcttc tagagaagca gccagtgagt gcccttaact gctgcagcat ctttctggct 55920 cttgtgaagg cattttatgg agtctgtaac accatatggg tatcaaaagc cgtgtggtca 55980 cctgatcttt gcagcaagac aggagagaga aggtgtttga gagacttgag ggatggtcta 56040 gacaccaaag aaaggtttgt gtgttgtgag caatgactga ttattaggtt gagattctag 56100 ccagggagtt tactatctct tgtttgattt attacatttt attgccatcc taggaattaa 56160 atagggttt acatttgcta agcaagtgtt ctctcaacat ccttatatcc cctccccgat 56220 ttctgacact tactatagct cagcttgtcc ttgaactcag caatcctgcc tccatagtgc 56280 cgggattatg gatgtttcca ctcccagctg agcctgttct tcatgtgttg catgggcatt 56340 gcagatctct cccattgctg tgaggctgaa ataaatgtgg atgaactttg tagtacatag 56400 tcgtttacat atcaagatgc ctttccttgg taaacagtgt tatgcttgcc tctcaaattg 56460 ggagtgtctt cctgttttaa gaataatcct cttccctcttt ttcttcctct ctcctcctt 56520 cccctccttc tgacagggtc tctttataga atattcctgc ctctctctgc cttccaaatg 56580 ttgtgtaggt gtacatcacc atgcctgcct agttagaatg acgtctcaga atgctggagg 56640 ttaatttcac attcttagtc cacattatcc tgactggtat gttcaaggtg ccctgacagt 56700 aggtacagac ccagcagacc tcaggaagtg accattacag caagatcctg tagctgctac 56760 tcatagcctg ttgggagccc gtgcatagga aagaatggca agaaaaaatg gctgccagag 56820 ggcgtcccca tagtatactc tgtcactaag catgcatgct tcagagcttg ccaaaacctc 56880 ctgagtccct ggcttggtcc ccaaataaag gtcattagag catgaagtct tggtcaactg 56940 attgagactc ctttggagag tgcaaggctt ctatgtagat gcccctgttg cctcctattc 57000 tgtctaattt cttactctcc ctgttgacag ctgccaatct cctttctcct aggactgtgc 57060 ccactcatca ctcactgtga taagacccct ttccttttct ttgttccttc ctggtctgcc 57120 tgcctgcccc cccccccacc tctccctttt catggtctca tgaaaactgc tctcagactc 57180 actatgtagc taaggatgac tggagctccc aatcctcctg cccctacttc cctaattctg 57240 ggattacaag tttatgccac cacattccac tttctgattt ccttccttac atcttaattc 57300 cactagcttt atcacactgc taaagctaaa actttctttg caaaatgaga tccagaaacc 57360 cacagactct ccatagtagg ttctgacagg gaaacagctg acattgtatg gtgttctgcc 57420 ttcccatttc tttgctgtgt gtgtttttgg acaagtctcg gtttcctgt atttccttcc 57480 ttccttcctt ccttccttcc ttctttttcc attttttatt tgaattataa acacgattgt 57540 tttacatgtt aatcccagtt ccctctccct cccctcctcc ctaccaccat ccccaactaa 57600 aaccctacct atcacatatc ctttctgctc cccagggagg gtgaggcctt ccataggggt 57660 cctcagggtc cgtcatatcc tttgggatag ggcctaggcc cacctccgtg tatcttggct 57720 cagggagtat ccctctatgt ggaatgggct cccaaagtcc acacctatgc taaggataag 57780 tactgctcta ctacaagagg ctccatggat ttctgaggtc tcctcactaa cacccacatt 57840 caggggtctg gatcagttcc atgctggttt cccagctatc agtctgggga ccaagagctc 57900 cctgttgttc aggtcagctg tttctgtggg tttcaccagc ctggtctgga cccctttgct 57960 cttcattcat ccttctctgc aactgtattc cagttcagtt tagtgtttag ctgtgggtgt 58020 ctgcttctac ttcttccagc tgctggatga aggctatagg atggcatata agtcagtcat 58080 caatgtcatt atcaggggag ggcatttaaa gcagcctctc ctctgttgct tagattgtta 58140 gttggtgtca tctttgtagc tctccaggca tttccctagt gcctgatttc tctgtaaacc 58200 taaaattttc cctctattat ggtatctctt atcttgtttt cttctattct tcccccaact 58260 caacctttct gctccctcat atactcatct tcccttctca ttctcctagc tccttcctcc 58320 ccttcccaat ttgctcagga gatctggtcc ctttcccctt ctccagggga ccatgtatgt 58380 ctctcttaga gtcctccttg ttacctagct tctctggctt tttttttttt tttaagattt 58440 atttattatg tgtacagtgt tctgcctgca ggtcagaaga gggcaccaga tcccattaca 58500 gatggttgtg agccaccatg tggttgctgg gaattgaact ccggaccttt ggaagagcag 58560 tcagtgctct taaccgctga gccatctctc cagccctttc taaaactgga cattagccta 58620 gcctcaaggg ttgtgatgct aacaatacaa gctaaagaga gcaatgggca tgatccaaag 58680 ccagatagtt cagcaaatat tgatttcatc ccttcccttt tcggaacaga ctccagccac 58740 accaaatatg taaaccagca agacaaaaaa cagcaggcta tcttttccca ctttttgctt 58800 tgtctatgtt tgttggacaa tgtcaaacta tgtagcccag accccttctg taactttcca 58860 tgtagaccag gctggcctcc aactcaaatt cccagcaatc tacctgtctc tgcctctgga 58920 gttctgggaa taaaggtgtg catcaccatg cctggcctca ataagtcctt gtcttatggt 58980 cttctttct cctcctttac ttcttatcct cctccctctc tcttcccttt ccctttatct 59040 ttattcttt attgtcaccg tttctttct gcttctctta ccatgcttta acaaccatca 59100 ggaagcaact aaccaaatgt tctcatcttg agagtcaggt cagaagatct aggagtcaag 59160 aaagacacgt gtaaataga gctcacatac caagcattag aaaactcgca gtggcactaa 59220 aagatggctc aggggataag aacacttaca tggtgactca caaccatttg taattccagt 59280 tgcagacaat tcaatatctc cttctgatct ctaagggcac ccagaatgta catacataca 59340 ttcaggtaaa acgttcatat acattaaaaa tataagcct taagaattag aaaagagggc 59400 tggagagatg gctcagaggt taagagcacc gactgctctt ccagaggtcc tgagttcaat 59460 tcccagcaac cacatggtgg ctcacaacca tctgtaatga gatctggtgc cctcttgg 59520 tgtgcagata tacatggaag ctgaatgttg tatacata 59558 <210> 2 <211> 60000 <212> DNA <213> Chinese hamster (Cricetulus griseus) <220> <221> misc_feature <222> (1)..(59559) <223> ERV insertion locus, allele 2 (ERV-c 109F not) Integration in allele 1, location 30020 (Locus of ERV insertion, allele 2 (without the ERV-C 109F)) integrated in allele 1, at position 30020)) <400> 2 tggttctatc gagacagcta catccgcttc tccacacagc ccttctccct gaagaacctg 60 gacaagtgag ctttccctcc acctcccttg actgcccagg ctagtgagga ggtcagcaga 120 caaacagaac agtgggctct gcgggcaggg aaggcaggct tttcgccgcc actcatcctc 180 tgccctgaag gagcttgcca agagggtgcc cttgggttac cgaaatgagc aaaacaagaa 240 ctcctgctaa tcaagggctt acatcctagc aatgagcttg agtaatcatg gctaacatgc 300 tagaaggtga tatgtgttag gaagaaaatg tagatctggg taatggcaga ttaagagaaa 360 cttgagagggg gaaaggcaac ttgcagtttg ctcttggggat gagaggaaac cttgaggaga 420 cctgggttca aattctatct ctgcagcatc cagattgtgt tcctctagag acatgaaaag 480 agacttaagc ctcagtttca gttagagccc agtggtccca tctgtaatcc cagcacttgg 540 gagtctgagt ctgaaggatt ttgaggtcat tctgggctat ataatgagac ccccactcaa 600 gacaaaacaa gccaggtgtg gtagctccta actgtaatcc tagaacttgg ctaaggcagg 660 agccaaaatc aagccaggta cggtattgga ggcccgtgat cccagtgctt agaaggtaga 720 ggcaggacaa tcagttcact gtcaccctca gctacatgcc aagttttatc taacctgggc 780 aacatgagac catttcatag aaaaagacca gcctccgttt ccacatctga agactggagc 840 agcaatacac tgtagctgct tatcatcaag tacttcacat gtgataagtt gttcctgctt 900 ggttatacca cagccacttc tcaatggctc acaatgaagt gagttattgc cattttaaaa 960 agagatgaga ggggcaaagg ttaaatgact tgcctgagat ctcatagttg atcatcggtg 1020 aagctgggat ttgaccatcc agggtcatcg tccccctgcc acacatagga atggtgaact 1080 gaaactaggc ccaaagcact tgatacttac tgtggcctgc aggcaacact ggagcattct 1140 gagaaatgac tagccctggc cgtgggtgaa tagggagtgg ggcagggagt gggggtgggg 1200 tggaggggtg cagcttctgt gcttctgtcc tgttgatctt ccagcagggg cccctctcag 1260 ttgagatccc ctagacggct gaccacaggg ttctgcctgc agccctgagg ttgggctcca 1320 gggctgagcc tgtgtctctg atccactcta gctctgtgca cctatgtaac aattccatcc 1380 agagatacct ggagacctcc tgtcaccggc accggatgct gccctcggac aacatgtggt 1440 ccagccagag gtttcaggcc cacctgcagg aaatgggtgc cccaaatgcc tggtctagtg 1500 tcattgtacc cggcatgaag gctgctgtga tccatgccct gcagacctcc caagacactg 1560 tgcagtgccg aaaggccagc tttgagctct atggggccga ctttgtgttt ggggaagact 1620 tccggccctg gttgattgag atcaatgcca gccccaccat ggcaccttcc acagctgtaa 1680 ctgcccgcct gtgtgctggt gtgcaagcag ataccctccg tgtggtcatt gaccggcgac 1740 tggaccgtac ctgtgacacg ggagcctttg agctcatcta taagcaggtg agatgtccca 1800 gcacctccca caggcaaccc tacagcaaag ccctggctgg ggtctgctgt gagacagagt 1860 tcaagactga ctctacacac ggggcatctt aacacagaca cgtcccactg gcctgtctcc 1920 tcatctgtgg aagatactgt cctttgagag ccattaatgc catgagtttg tagtcatagg 1980 tagtattttc agaggccctg ggacctgctc agtttccatg gtaattccaa gcctctaggt 2040 agtaaccta ttctctcatc tgtaaagtaa gactgtacct ggctcctcct ttgtgtgctg 2100 tgagaatggg tggttgatc ttcacacaag ggtttgctaa agtaccaagc tggaggacat 2160 agaggatatg aggccatgaa cctcaaggcc tgcctatcaa gagttaatta ttagtgtcta 2220 tcattgtat aacgattatt atgttactca tcttcttcca aaaacagatt tgatcttgct 2280 tatttataat aagtatagaa ttgcttgggg tttttgtttt tgtttttgtg ggttttgttg 2340 agatagggtt tttctgtata gctttggatc ctgttctgga acttgctctg tagaccaggc 2400 tggccttgaa ctcaccttg actgcctctg cctcccgagt gctgggacta aaggtgtgca 2460 ccaccattgc ccgacctaga attgtttttt attcagccaa aaaacattac cttgccctcg 2520 tggtctaatt ctctctagaa acacccttag gagcacacca ggggcatgcc ctatagaaat 2580 cctaggtttt ctcagtccag tctagttgac aactgatatt agccaccaca gctggacatg 2640 gggctccacc ttccacctcc cagtatttgg aaggctgaga caggggatca cttttagttc 2700 aagggaagct tgggttacac agtgagttcc aggctagcct gggcttcaca gtgagaccct 2760 acttaaaaac atcatacaaa caaacaaaca aacaaacctt taaacgtatg tttttaaggg 2820 tttctgaatt ctgaggacag tttttaagat ctttgagcta agattcacca aggtctaggt 2880 ttgctacagg aagggaaaac actgatcacc tgacctgggt ggcctcatgt ttcctacagg 2940 tcctgatcac cttagaataa tgtgctggca aagggttccg tctttgttag ggatgccatc 3000 actaggtgtc cagggtggaa tccaggcctg cgtttttagc atgtgcagtt ctactgagct 3060 acacctccag cctaaaaaat gtaaaggagg agatgcatgc aggtgtgcat acacctgtaa 3120 tccctgggca acagaagcaa gaggactgtc acaggttcac caccacctgg ttacagggtt 3180 tataataagg tcctgtcaca aacaatgtag tcttggaaga gaggagagga aatggggaag 3240 aggaagtaat ttgcagttta aaatagagca aaattgccgg gcgttggtgg tgcacacctt 3300 taatcccagc actcgggagg cagaggcagg cgcatctctg tgaattcgag actagcatgg 3360 tctacaagag ctagtttcag gacagcctcc agagctacag agacaccctg tctcaaaaaa 3420 aaaaaaaaaa aaaaaaaaaa agcgaaattt gtatggacat ggcaacacgt gcctgtaatc 3480 ccaacactta gggggctgag gttggaggat caggagttca aagttatcct tggctgtatg 3540 tgagcttgaa gccaacctgg gcctacagca ctgtctgttc cacaatctgt ctttatctgt 3600 ttgtctcctt atgttgttca gctcggtctg ttctctgaat gtttgtctca gaacaaacaa 3660 aattgaatag ggctgggcat acgcactttt caaatgtcct ttgtccccca ggtatctttc 3720 attgctgatt tggttttgag cttgagggtc agtgtacaac aaggtggtta caatggtggc 3780 tttgtctgtc tctgatctcc tttatctagg acagtgccac tgctgtatcc ctggcaccct 3840 ggtcctttgc aggccaggca ggccagcctg caccaccgcc ccacactgtt ttatctgttt 3900 ttctccttat gttgttcaga tcggtctgtt ctctgaatgc cctgtaaact agaagatagg 3960 ctaacaagct gatggggttc agggttgaga tttttggcaa aaaacactca tgtgatgcta 4020 ggtacctcat gtgactgtca caaggcacaa ccaggaatct ctagttccca atgccgaatt 4080 tgaccttaga ctaaggtggc tgccaccaga gccacatcct cccctttgta gcttttttca 4140 tttttcttta cattatttat tgtgtttgag tttgtacatg tgtgtggtca cacatgccac 4200 agcacacatg tgggagtcag agggcaactt atgggagttg gttctctcct cccatcccat 4260 gggtcctggg gcttgaactc agcaagtacc ttataagcta tctcaatact gtttgcctcc 4320 aaaatgtatt actttgtgta gctgtccctt ttctgttgct aataacagaa tactacagac 4380 agtgtaagtt acaaagaaaa taggctcaat tgtatccatc ttttgctgcc catggcatga 4440 aaacacttgc atcccaacac aggccagagg tcgctttagt gtgaagcagg attgagtgca 4500 tgtctgcatg gctaagactc tctacttctc tggtttcctg atccctaggg ctctgggact 4560 ctagatcctc tggacccctt gataatagag ggagcttcct gtttctcaga agtgcctttt 4620 gaccatatat cccagaaact gattccatcc atctctgccg tgtgtcacta gtcactagat 4680 ggcgtcactg tcatctctca ctagtgtctg agatggcctt gtcttctctc ctgcccacag 4740 cctgctgtgg aggtgcccca gtacgtgggg atccggctca tggtggaggg ctctaccatc 4800 aagaagccca tagcagcttg tcatcggcgg acagcggtcc gctcatcact ccctcatctg 4860 ctggcccagc aaggctgtgg ggaaggcaag gactcaggac cccctatcca caggtcagct 4920 tctaggaaag atgctggggc caggagcctg ggacacactg agaagccaga ctctgcggcc 4980 accacctcag tccccggaaa ggggaagaaa ggcaaggcaa aaagtgccac agccctggtc 5040 tgcatcaccc tgcagaaatg ggagtcccac aacaccaggg tgggccccac cttcaacagg 5100 ttaatgtgtc tgaaacagcc tgaggcctgg ggtagtacca tgtcccccaa accccgcagt 5160 gttcccaagg ccatttctgc ctgctctcca agccctcccc aagcatctgg gcttgccctc 5220 ctgccaaaag gccaccagtg atagcaagta tgaaccaaat atctttaaat acataaccaa 5280 atgagtatta caaagtagtc accctgccag gcagttagac caaaggctcg gtcctagagt 5340 gcgcgcccag agtccagacc catgctgctg ctctagccag cctttgccct cacctttctc 5400 tggagaaagg tgctgccacc atgcccttcc ccattcctaa ccagccccct cagccctcat 5460 aacgccctag tgaggtaggt gctattgtcc ccattttcca gccgaggtag cagcaagttt 5520 aaggacgttg cccgaggttg cacagctcag aaggggcaga gctgggatgc agacccaggt 5580 ctgttggtct cccaaccctg tgttcttccc actgcctctg gaggaggagc tgggaggggc 5640 tccatctgcc cttaccttgt atcccccacc tttacacatg tactgtggaa caattggtca 5700 ggctggggcc tcacccagat cctcacagct tcccttctcc cacagccccg tcctacctcc 5760 ctagtctcca ttccaaggcc tggctgcctt cttcccatgt gctccgaccc cagggccggg 5820 tcctcagact accgaatggc caactggtgg gctctaaggc tctgtcaacc acaggcaagg 5880 ccttgatgac tctacctact gccaaggttc tgatgtcctt cccacctcac cctgatctca 5940 agctggcacc cagcatgctg aagccaggaa aggtgggcct cgagctgtgc ctcacaccct 6000 ggcgggtagt gctgagcagt gggatcgggg ctgaagggca cgaacagagg gcagcgctcg 6060 gaccatacag cgccccaggg aagggcttgt cttctccaga accctgttcc aagacagagg 6120 cctgatcata tctctttccc tcccctcctt gcaccgaggc tgctattccc ctgcaccttc 6180 gaggccccca ctttggaagt gcctcgaggc ctctgcctt tgaagttgga cctctccta 6240 gcaccacag gaaagtcacg gccaaggca agttcaaggc catactctgc gawaagcca 6300 gggctgaggc attackccaag aagaggctga gcctccccaa acccttgacc cttattctga 6360 catgccggac actgagaacc atggggta ggaggctaga gaaacccctg ctctgatctc 6420 tactgcccca tcctggatcc agcatcaat taaaaaagc aattaaagtt ctctggactt 6480 ggcttgaata atgtgcggct aggctcataa aagagttgac cagcagggcc tccatcagca 6540 agggccacag tcccaccca gcgacagaca ttggctttct ctgcagggag acggatggtt 6600 ggggaaagag ccttcactat acgatgatg acactgagac atggcttgcc tgagaccaca 6660 gcaggcggaa agtcagccat caggagtgcc ccttcccaa vakaagctgg gctggcagaa 6720 gagcttctga tggtcacaga acatgac agagacggg ctttccctaa acctcagcct 6780 ttctccggag agtatgtccc tcagtgaggg gtcggtcaag acacctcaa ctgcaaacc 6840 CAagaaacag gtcaagtgtg gcattccta cctgtagagc ctcagctgct ggtcctccaa 6900 aagcggtatc taggcttagc gtgtgaatgc ttcctgagtg gggcagggtg gaggaggag 6960 tgcggtgttg aaatccaatg acctgtgtct cccaaagtca gaagagctca tgcctgcaca 7020 gtggtgtgtg tctgtcctcc caggacttgg gaggcagagg caggtgcatc tctgaattcc 7080 agtccagcca gggatatacc aagaggccca gcctcaaaag caaacaaacc tcctcgtgac 7140 caggagatca gcagatgcca cctccagacc tggccactgt tcacttgagc agagagcaca 7200 gtccctggtc aacacttgct ttctcagcag atcctcaaaa gcagacttgt gagtaggac 7260 ttttattatt ttaagtccag agagcggtca cctgcccaac gccacacagt aagtgagtgg 7320 tttaactggg atttatctta ggttggtagg actaagggt caaagacctt gaccgtactc 7380 agcaccaagc ccactgtcct tgagctgggt cacggccctt ctttctattt tccaattggg 7440 ggttaagcac tgtctatcgg tgagagagcc gcaggcactg cagggtccga gagagatcg 7500 aggtagggg tgccacaagt tcactagggg ggtccctgga tctggtgcct gggaggagga 7560 ggcggtgcaa gtgcaggtgc aagggcatct gggccacctg ggaggacgc aggcgaaggc 7620 gtctgaggag agcttcgtcc agcacctgga gtgggaaaga cagcagccca cctgagttcc 7680 agccagagga gcccctggct ctgatggacc tctctggtct gcaactgcca tcattttctc 7740 aacaggcagg cagggatttc tctccacaca gagctaagtt acgtttcagc tccttttgtg 7800 tttagtgaag ccatgtgatt aagccactca ccaatgggat gtgaggaaaa cacagaccta 7860 gccctcaaag ccccgattca caaaaacatc cagtctcccc ttatggccag gaagcaatag 7920 cccttttctc caagtggcta cccagagtca ggtccactcc tactgtatgc ccatttgcca 7980 gacttaatcc aagaagaaac aatggacttc agggcctgtc agaggtcagc tccccactcc 8040 ttgagctaac agacagaagg aagcctgagg aagtcacagc accacagagg caagggtggc 8100 cccagaggcc aagcctgttc ccccaattcc ctaaatacag agcaggcatg tgggcctgaa 8160 gacctacctg tctctggagc ttggtggtct tggctggcca cagaaagcgc tggcccagaa 8220 cggtgcccac tcgaggagca taggtgtggt ccccaagcac tgggcagagc tgtagagcca 8280 tgtgcacctg aagctgactg gggaacactg aaagatgggg ggaggcagtt gctcttcttg 8340 atcatcaagg actgtcccca ttcccagagc ctgtgcatct atgaggtgac ctctgcctcc 8400 acagagggcc atgagcagat caggacaatg acagctggca accttcccag cctgggggca 8460 ttgaacccaa cagaactgaa gctcctggga ataagtatgt gggttgggtg ctaaactgtg 8520 ggtgtgcacc taaaactctg cctcctgctg agcccactct cagagtcccc agatgctctt 8580 ttttttttgt tttgtttttt gttttttcag gcggggtttc tctgtggctt tggaggctgt 8640 cctggaacta gctcttgtag accaggctgg tctcgaactc agagagaggt gcctgcctct 8700 gcctccccag tgctgggatt aaaggcatgc cccaccaccg cccggccacc agatgctctt 8760 tggatgttgc aaaacaacat cttcctcata ggcttcattt cccctgcaga gtactgttca 8820 gccctacctg tcagtggctg cagctggacc agagcacagc cacagcctgt ggccatcaca 8880 tggaaatggc tgagggtcct cttgacacct tctaggatgt cctttcgaga tggggatgtc 8940 acggggatgg cctaggatac cagtcaagaa tgacttagca cacacgtagg agcccaattg 9000 agccacccct gctgccggct gacttaggac agggcctcag aacaggcagc agcaacttgc 9060 ctgttagagg acaggggaat agccacccca tgcacatagt gcagactcct ctgtaaggca 9120 aaacctgaaa ggactcctag tggcttctga ctcacaagat caatgccatc catgcgttcc 9180 agcttcaggg ccacgtggat agtcccctca gaaggctcag ggatgccatc agtgatgcca 9240 ctgagaaaga gaaaggaagt ctcagagcag ccagctggga ccttccttgg ccctgggagt 9300 caccccgcat ctctcaccag taggtggctg tgggcctctg tgttctcctt gagtgaacga 9360 agaacttctg caagcggctt gctgtctggg ggcagctgga gagaagcaca agcccagacg 9420 cctccctgaa agaagaggac aagtcacata aagcctcttg gttggaaaaa atgagggcca 9480 gattggctgc tcctgcagag gactagcact cggcacccac atagtgggtc ccaactgtaa 9540 ttccacttcc aagagagctg atgccctctt ctggcctctg gggcaccggg catgcactgt 9600 ggtacacaga cacacatgca ggctggcctc cgggggcaca aggcatgtgt ggtgcacgga 9660 catacatgca ggctaaacac acatttaaaa acaaatcttt ggtcttttttt aaaggagacc 9720 ctcccaccaa ggggctggag aaatgaatca gtggttaaga gcactagctg ctcttccaga 9780 gtacctgggt tcagcacca catggcagct cacactgta gctccagttc cagggatct 9840 gagaccctca cacagacaca catgcaggta aaacaccaat gcactatata tatatatata 9900 taggataga tagataga tagataga gagagaga gagaccac cttcccacct 9960 ccccaaatga acaccaggac tacagtccag acctaagaaa caaatcctgg ccaggcagtg 10020 gtggtgcacg cctttaatcc cagcacttgg gaggcagagg caggcgcatc tctgtgagtt 10080 cgagaccaac ttggtctaca agagctagtt ccagggcagc ctccaaagct acagagaaac 10140 cctgtctcaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaagggggg 10200 ggaataaac aaatcccaag accttttttc tggcccccag agactcaga CAATGCCct 10260 aagagttact accatgcca ggcacctgaa taggactt atacagtg ggtgtaaaat 10320 aaagcccac cacactccctc acttacttc caggtgctcg cacaacctgg agctcccggt 10380 gtttgagcccc cagggcctgg ctcagctgtg gcagcacaga aagcaggtc agctccccag 10440 gtctccctga gaagtaagg ggagaaaaca agttgccaga gcaccatcca acgaactcct 10500 ccccaccagc ctttccctcc ttgtccctgc ccatacctgt cacaggcaga ccctgtggct 10560 tgttcagtgt caccagaggt cctgaaatat tcggtagatg tcagcaaaag cagggcagcc 10620 tgtgtgcatg cagcctctgg ttataagctc ccaagcccac tccaaactga acttcctgct 10680 ccccgcaccc agggcctttg cacctgctgc cctgagcttt cattaatatt gggtccctta 10740 tcacacacct ccctataatg tgtgcccctc ctacagtcct tgactgtttg gtctgagacc 10800 agctcaaaca ggccggcctc aaactcaact ataggtaagt ctggtcttga actcttgatc 10860 ctccttgcct ctgtctccca agttctggga ttacaggtgt ggtcaccact cccagaaaca 10920 gcagagattc actattcctc acatcagaac gcttgctctc tcaaggcagg agccagccat 10980 atttatccca gtactgtgta gcctcctcat tgctacaatg cactgaagtt cagcaaagtt 11040 gtcattccta aaggcacaca gcatgctgct tacagacccc aggaaactaa cttctgctcc 11100 cgagggtctt agaatgcgag gcggtgtgaa ggtttcctat gcctggcgga gggaatgtga 11160 cggctacagg ggtccgactc cagggggtcg gaggctccct caccttgctg atccaccaca 11220 gctgccctca gcacctcagc cagctcctcc gggccgaggt tctctgttcg cagaagcccg 11280 gggaagggct ggtcctccac tgcgtccttg cacttgctgg aaccttgagg ttgaggccga 11340 cccctgagaa aaacgacaga agccggtacc tccaacgccc acgctgccca gtgcagcctt 11400 ttcgtacaca accctcagaa cccccggctc ggctgacagt tgtggtccac aggcccccaa 11460 gactacagaa ggggcatcag acgctcggag cgccaaccat ctcgtgtcgg gtcacacaac 11520 gcggctggga ttagaaccca tgtcctgatc tgagtgcccg acctgccctc cccttgtcct 11580 agcctccctc ctacacggaa gtgttgagtt ataatcaccg ggcctcggtg ccgaagcctg 11640 catctctcgg ccgtgcgcca gccgccagcc ggggccgcca ggtgccagac acacaacgcc 11700 aaacgcggaa gacgcccatc gccgcagcaa gcacagacgc atgcgtgtcc acggactgcc 11760 ccaccgcgtg catcctccgt gcgccgattg gtcagcctgg ctgtcaatca gagcatcgag 11820 caggcggagc ttcgaggacg gaagagccaa actttccttt ggctgaggaa ggtaaagtac 11880 ggtctccggc tctgctttgg gggaaactga ggcaggaggc gcggttatt actcgcggta 11940 gaggacgtgt cttagaatag aaagaggcga gtttgcagat agattttc agttctcagg 12000 acccttaatc tgaaacccat ccttgtaaag cagagggaga aggtgaagcc ccacagcctt 12060 cgggccaggg cacctgcatg gaatggtct gcgacattaa ccatcccatg actgtggctc 12120 aggtggtaga gagatttttc taggatgcac gagttcctgg gttcgatccc cagcaccaca 12180 cgaagaccaa gatcacagat acagactgag ttggagacca gcctgggata tgtaaggccc 12240 ttctatcaca agaaaaaaaa actaggccgg gtgtgtggt gctctctc aacagaggcc 12300 aacctggtct agacagtgag ttcaggaca gccggagcta cacagagaaa cctgtcccc 12360 cccccccaaa aaaaaaagaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa ur lav la la la la ver back the higher the longer the longer the longer the other, the higher the other 12420 cactggccag tatgttgtta attccaaaga tggaatgagt gtaggaaaa aggccagcca 12480 aagaaacaca ctttggctct gcaaccagaa ccaagaat gcaccttgt cagaagccag 12540 ccttggtgac gcccccccccc ccaatgccta gctagccgcc agctagccta gcatcccacc 12600 cctcaggttc caggacctgg cctgagaagc aactaacacc ttcccccatt tccttagtta 12660 12720 tctttctcca cacaggagat tccggatacc tgcctgagag caattaacat tcattcctcc 12780 cccacctcct tttctctgc ttcctccttt ctctataaaa accccttgta ataatcaata 12840 aatgggcctt gacaagaaaa cctgcttggt cccgctcttt ctttcccgcc catcattttc 12900 aggcgtgatc cacctcggac tcaggaatta ccggagcccc gagggccggg gtacaccctg 12960 accctgaaac cagtgccaga gaacacact ttggccctgg aatcagagcc agtgaaagaa 13020 atacaccctg gctctaaaaat cacagtcaaa aggctaaaaa ggaccagtga aaaggaacaca 13080 ctttggccct gaaaccagag ccaaatctaa ccctaaacct gtgcagaaaa ccaaccaatc 13140 cctgagcagg agatctagcc attctgagct cagggattga aatctgacca atccccaccc 13200 tggaaatctc cctgggaaaa ctctgccccc ccctaagaat ccctatataa accctgtgcc 13260 tgttcagctt caggctgcct ggctgccctc tgccactgga ttcttgaatg aggtttgggt 13320 aagaactttc gccggagcag agaccaccct acacactggc tctccagggg actggagttg 13380 aactgttaca atttaaccag ggaaaccctc tcctccccaa gcagagctga tacactgagg 13440 aaggcctttc cctgaagcag ggctgtaagc cgctatgctt actgtgacct gtggcattcc 13500 ttggctccta aacgccagaa tacctttcca tcccagctgt aacactcagt attctgtgac 13560 tccggagtgc cagaatactt tgcatctgag ccataacaca gtattctttg gctcccgagt 13620 accagaatcc attccttccc atccaatccg ttgaaatgct tacaatgaga aaaaccacga 13680 acccaaatca ttgagaccaa attaattaaa gttttttttt cattcatgta cgagactgcc 13740 tcccccaagg cctgatttga gaggtcagca ttggatgtga ggaagacaag ggggttttgt 13800 tgttttggtt ttttgagaca gggtttctct gtgtagcttt ggagcctgtc ctggaacttg 13860 ccctgtagat taggttggcc tagaactctc agagatctgc ctgcctctgc cacttgagtg 13920 ctaggattaa aggcatgtgc caccaccgcc tggcctcacc tggatcacac agacacatat 13980 atacataatt aataaaatac agacctggcc tttaatccca gcactaatct acatagtgag 14040 ttgcaggaca accagagtta catgaccctg tctcaaaaaa aaaaaaaaa aaaaaaaaa 14100 acaacctaaa taaataaaat gcagaaagta acaatatgta cataataaaa ataatgaaat 14160 aataacataa ttgaggaaca gtggggcatg gtggcatgtg cctttaatcc catcacttac 14220 tgatctacat cccaagcctg aaggaccttg ctgaaagggc tccagcacta gactgttgct 14280 tctggcagag aaagtggctc tcaacctgct ggtcaacgac ccctttgggg gtagaagggc 14340 cctttcacag gggtagccta agaccatcag aaaatacaga tattcacatt acaattcata 14400 acagtagcaa aattacagtt ataaggtagc aaaaaata attctatggt taggagtggg 14460 tctccataac atgaggaact gtattaaagg ttgcagcatt aggaaggttg agaaccacca 14520 atttaggggc agatgctgcc tccttcacag cactcagcca cagttgtgtt gtgtccttgt 14580 tgaggttcta tccggtcctc caaagttcag ctgtgggaaa ctaaatcttt aacttcataa 14640 taatggtaat tggaggctgg agtaattagt accagataaa atcatgaaga gtgtgcttcc 14700 atgatggcca ttaacagctt tctaaggaga gcagagaaaa aaaccttttc ctcgttgtgg 14760 aatgacttcc tccgtgttgg tgtgcaccag aaaggccccc tgcttgtgct aagtggagct 14820 atgccatgct cttggactct gcagcctcta gaaccatgat ccagatcaac tgtgtgtctg 14880 tgtgatttgt tttattttta ttatgtgtgc atgcctgtgt gtctgcgtaa gtgtatacca 14940 tatgtgttgc aggtgcacgg agaggccata aaaggggcat cagatcctcc gaagcttagag 15000 ttttaggcag tcgagagcct ccacgtgggt gctgggaact gaactcagag cctctgcaag 15060 ctcagccagt gctcagccag tgctcctcag tttgtgtgct tgttttcagt actgcagatg 15120 gatccaggac cccacacgtg ccaggcaagc attctctcac tgagctgcac cctcagactg 15180 gtctcaggtg gtttgtata gcaacagaaa acagacatgt ggaaggagtg atggcgtatc 15240 ttgactgaga ggaggaataa cttggctact cgcacagcac atgtctgggc aggttcacaa 15300 ggaggcttcc agggacaatt ggcacctaag aaccctgacc taatgaatgg atgaaccatt 15360 cagaatttgg atgggttctt gggggggggt gagggaactg tggtgggtgg ggactgcttg 15420 gaggaagtag ggtgtttgtt gtgggtggct ctcggaggct atgtcttaca ttggctcctt 15480 tctgttttgg ttctttcaat ttcctgttgt gtgctattct gctacaaccg cctcacccag 15540 tagattgcag cctctgttaa tcaagacagg gtcttactgt gtagccctgg ctggcctgga 15600 actcatagag atctgtttct tctgcctctt gagtgctgga attaaagacg tgtgccacca 15660 ccaccacctg acctagagga caactttatt ttttttaatt ttttatttat tatgtacaca 15720 ttgttcctgc ctgcgcacca gaagagggca ccagatctca ttatacatgg ctgtgagcca 15780 ccatgtgggt gctggtactt gaactcagga cctctggaag agcagccggt gctcttaacc 15840 tctgagccat ctctccagcc ccctggatga ttaattgtag aagtttcagt tctttctttt 15900 tgtttttgtt ttttgagaca gagtcacact acgtatcttc aactgatctg gaacatgcta 15960 cgtagacaag gctggcttca aatgtgtgtc aatgttcctg cctctgcttc cagagtgctg 16020 ggattatagg catgtcccta aatctgttat ttttttaaac acttattttt gagggggagt 16080 cggtggtggt gctgttgtac gtacatatgt agagatcaaa gggcatcttg tgggagtctg 16140 ttctctcctt ccaccaatgg gttctaggga ctgaattcag gttctggagc ttgacagcaa 16200 gcaccttaat ctgctacaac atcttgttgg tactaaatcc atggagaatt gaaaggattg 16260 tttattgttg ggcccgggaa tatagctctg tggtagtaga atgcttgcct aatatgtgtg 16320 agattcttaa aaaatcattg attgttttct taaaccaact ttttaatttg ttaaaaccaa 16380 cttttaaagg atatatctag gcatggtggc acacagcatt aaccctagca cttgagagat 16440 agaggcaggc agatctccta tgaatttgag gccatcctat tctacgagtt ccagtacagc 16500 caggactaca ggactgtgtg atatgcacaa cacaggaagg actgatgaca aatttggtaa 16560 aaaaaagaaa gaaaaacagc aagtggtttc acattgtccg gcttcacctg gagtgggtga 16620 ctgtgggtac tcctgaagcc tcccaggagt gggtgactat tctgtcagac tgtgggtatt 16680 cccgaagcct cctaggaggc ctcctgtgca tcacagaagt ggcttgagca agaacttggg 16740 acagactggg caagtgcaca gctataatcc cagcactgca gaggtgaggg ctgatgggtc 16800 aggagctcac agttatcctt gactacagga agtctgaggc cagtctgagc tatgtgagac 16860 tctaaaacat acatgagtca gtgagttggc tcagccagta agggtgcttg ataccaagtc 16920 tgaccacctg agtctgatcc ccagaatcca catggtagga gcagagaacc aactcccagg 16980 agctgtcctt tgacctctgg acttatgcta ttgcatatgc ataccaatac ataactccct 17040 aaaatacaaa atacatatgt ttgggctttt tgtttgtttt gttttgtttt ttcgagacag 17100 gttctctgt gtagctttgg agcctatcct ggcactcgct ctggacacca ggctggcctc 17160 caactcacag agatctgcct gcctctgcct cccgagtgct gggattaaag gcgtgtgcca 17220 ccaacgcccg actgatgttt gggcttttt gatggtttt atgtgtgtgt gtgtgtgtgt 17280 gtgtgtgtgt gtgtgtaatt agaaggagaa agagagagag ggagagagag acagagagac 17340 agagacagag acagagacag agacagagac agagagagga gagagagcca aagtctggtt 17400 tgtggcccat tcaactaagc agacttctcc tccaccaatg gtgtccacca atggcccagg 17460 aaaggccatt tccccacttg aaccagcgcc tggccctctg gagggttagt tcctttactc 17520 tttggaggtc tccctccctg ccccatagca ggctgccctc tcttgctccc ttcccacagg 17580 cagctacctt catcccagtg ggctatgagc tcctggaaga aaaggaggag ccaaattttg 17640 cctaggctga gggctcagtg ccaggtggca gccacagtca actgctgaac ttgtgaaact 17700 ggaccccagg agaagaagcc tgggacaaac atcagcttgc atcagctccc tctggctcag 17760 ttacagcttt gcagcaggtg tggacaggct ggtgcttcag caatgggaaa caggactgat 17820 gcaaaccagg cctccaggag gccaagccct ggagccagcc tgcagtgaac cagcctccag 17880 ccacatctac aactgtgtga gggcctagaa tagaaagtgt acttgacctg tctgtccttg 17940 agtgctcttc tcttctcaca cttggctcca ggcatggggc agcttccagc gatggctttc 18000 cagacaagct tgagtaactt ggccctttgt cttagtgttt tctaaatcca aataggcttt 18060 acagggagga ttgtgaaatt atgttagctt tggatctgag agccagactg aaacctggct 18120 tcctacaggc ctagtctttc ctgcttaggt cagttacctg tcactaataa atgggggctc 18180 cttttcctac cagccaccaa gatggctgaa gtggaggaga agaaatgaac cttccacaaa 18240 ttcacatact acaatgtgga cctggatcag ctgttgacag gttctaggaa ctgctgatgc 18300 agttgtacag caccccagag gtggtgtctg aaccacggcc tgtggcagaa gcagtactcg 18360 ctactcaaac acctgagaaa gctcaagaag gaggcaccaa ccatggagta acccgaggtg 18420 ctgaagaccc acctgaggga cacgatcatc ctgcctgaga tggtgtgtgt gtacaatggc 18480 aaaaccttca gccaggtgga aatcaaacca gagctgatca actgctacct aggcgagctc 18540 ttcatcacct ccaagcccat gaagcatggc cagcctggta ttggtgccac ccactcctcc 18600 agctgcatcc ccctcaagta gctgtggcca acaaagactc atgtttaaaa agaaaattgg 18660 aagccaggca ttggtggcac acgcctttaa tcccagcact cgggagacag aggcaggtgg 18720 atctctgtga gttggaggcc agcctggtct ccagagcgag tgccaggata ggctccaaag 18780 ctacacagag aaaccctgtc tcgaaaaacc aaaaataaat aaataaataa ataaataaa 18840 taaagaaaag aaaagaaaag aaaaatgggg gctcactctg agaagacctg actcctcctc 18900 ctcctcagaa ctctctggtt tgtttttgca gtgctggggt cactgtgcca ggctagagac 18960 tcactgcaat ctgagggtgg aagtgctgag caggaacagg gaactgtgta taagctggca 19020 taggcattga taaattcccc tgtagatgga ccaggacctt tcaacccgaa cacatggaaa 19080 gttattgtaa aatacaacag ttgggaatca aaagagtggt ttgatccacc ttacaggcaa 19140 atttcattcg gaaactgaca acacatatgg atcatgtggt ctgcctgaat atcaaatatg 19200 gcaggcctca aaaatcaact gcagatttta atataacatt taattattca tctattaatt 19260 aatttattct gtctcataat atcaaacctt atctattaaa agggagcagg gctggggatg 19320 tggctcagtt ggtagagaat ttgcctagca tgctggaaac cctgggttcc gggttcaatc 19380 cccagagcca cataaattgg atgtggtgtt tcacatctgt aatgctagca cttaggaagt 19440 ggaaacaaat ggatcataag ttcaaggtca tcctccacta cataataaat ttggagccag 19500 cctaggcttc tgtatctaga agaaaaaaca gggcaccact gatgcaactc attgcataaa 19560 agcaattgct gtgtgagcct gacaatccaa gctcaatcct tagaaccaag agtggaatga 19620 aagttgtctt ctgggccatg cacgccttg atcccagcac tcgggaggca gaggcaagtg 19680 gatctctgtg agttcgaggc cagcctggtc tacagagtga gttccaggat aggctccaaa 19740 gctacacaga gaaaccctgt ctcaaaaaaa aaaaaaaaa aaaccaacaa aaaaaagaag 19800 gttgtcctct gacctccaca cctgcaccat agtactggca taccaacaca cacacacaca 19860 cacacaca cacacacacc agtaatgaca aatgctatct cctgtaattc tcggcaaata 19920 gtttcaaaca aagaacacag gcaaagatga ggtattggca taaataattt acttcaggct 19980 ctaataccat acattagtgg ggaatgagaa caaaaggagg aattgtctga cttcccctgg 20040 ggatcacaaa ctctatccat tcggcccagc aagggcgcca atgaaaggtg aagaagccac 20100 gattccatcc aagtccagct tggtgaaccg gtgagtttac taggttactt acaggtgggg 20160 cctgggtgac tcaaaatcac aggtgactcc ctccaaatct gcatcagtga catcctggct 20220 ttagttaacc ttttacctct tatatactct agcaccgccc caagatcatg agcagctggg 20280 gcggggcagg agggaggaat ggctgggatt tcaggtgcta agaccccctg acactctcct 20340 ccctttctaa tacaggtgtt aactatttcc ataccctagc cataggcctc accgtcactg 20400 tggcatttgg ttcattttgt tgtcttgatt caggtcaaag tattctggag gccaccatag 20460 caatgtctgt tgcttgatga gcatggtcaa ggcaggaggg gactgcagag agggagtggc 20520 acatagggat ggcatgtgaa gaccgagtgg cagacttggt gccaaagcct gccagggaca 20580 agtgtttgtc ccctaaacta cctgtgacat gaaggaagga actgagccag gctaggaagt 20640 tctcccgtga ccagcccacc ccagagctcc agcccttcct gcagtttcct tggtgctgtg 20700 ctgtgagagg ggtgcaggtt ggtgagaagc tctccagctg ccaggcttgt gggtgcttct 20760 agcagagtgc aaggtctcca gtcacatcct tggctgggga cggcatctga ggacttgggc 20820 cttcattgca gcatcttcag acaggcgggg aggagggagg aggtcttggc cttggacggc 20880 tgttcttcct cagtggcatc caggaactgc tgcatataac tggaggagcc aagcagcatg tggcctgtgg cctgcatgct gaagagggcc cagcggagca tttccctggg cagacagcag gcactcaggt taaacactcc ccatgctatc tccccaaatg tcttctccat ttttctatgc tggcctaaag cagttggctt caagttagac ttctttcttt gtttctactt cttttatttt tgagatagaa tcttaacact ttatctcagg ttagctagaa attcatttat gtagctcagt ctggctttga actcacagaa atctcctgcc tcaatctcct gatgctgttg cagttatggg ccaccacacc taactttttt tggttttggtt tggttttgat ttggctcttt ttgaaacaga atctggagat cctcctgtct tgtcctccca agtgctaggt attackcat gtcccaccat 21360 actgggctca agctagcttt tctccttagg aagatttaag tgtcactaaa gtaaaatgac aaaaatgctt ccaagtacag acaggaactg ggaaccaggc tcatagctag ccatccctta 21540. cacatagttc tgcctctact cttgtgacaa cttttctgga accccttgct ttttcccatg cttaacttga gttctcaaaa caaagcttt tattttcttg taatgatatt ttgtttcttt 21600 cttttctctt ctatggtgct ggggatggaa cccagggctt tatttgtgca taagcagctg 21660 acctgccatg gagctatgtc cccagcccca tatattttt ttttcaagac agggtttctc 21720 tgtgtagctt tggagcctat cctggcactc actctggaga ctaggctggc ctcaaactca 21780 cagagatccg cctgcctctg cctcccgagt gctgggatta aaggcgtgtg ccaccaacgc 21840 tcggcatcca gccccatatt ttttaacta ctctggacca gccagattaa tttacataac 21900 acatgtctac cctttgtaac actgcttctt agactttata gtttcccag gccagttctc 21960 agaatgctgg ccacaaggct cccatgcctg atatcatatt ctaagcatag aacttggacc 22020 agacagggct gaacactgat ccagagtggt ttgtttatt ctaaaatatt taaaatatat 22080 tttaaaatat tataaagatg ggtgatgtaa ctaatttagt gcttgccttt catgaacaaa 22140 gccctgggtt tgatccccag caccgcaata acccagagtg gtaatatagg actgtaaacc 22200 taggatccag cactgtggag gtagaagcag gtggatccca agttcaagt catccttggc 22260 tacatagcaa gtgtgagacc agcctgagat acatgagacc ctgccaaaa aaaaaaaaaa 22320 aagctttaac tgttctggtt ctgagctcca gcagccagaa gctttgtgac ttgtaaaatg 22380 ggtaatgagt ttgaactaa tgtgaagcac ttagcattg gtctgatgga cagaaaatgg 22440 gatagcagc aagagcagc tggctgaatg caactctctcc cacagctatt gatagggct 22500 cggcaggggt cactaggagt tgctggtctt aagaacaata aggagaa aacacagaaa 22560 agagaaatac tgccaggccg cggtggcaca tgctttaat cccagcactc aggaagtaga 22620 gabaggcaaa tctctgtgag tcgaggcca gcctagtctt cagagcaagt tccaggacag 22680 ctacggttac atagtgatac cctgtctcaa aaagcatat atatatatat atatatat 22740 atatatat atatattgaa tttagtggcc tgaggcaatg gctcagtggg taaagtgctt 22800 gccatatggc catgaggccc tgaagccca tgtaaaactg gggatggctg cctgtatctg 22860 tgaccccagt actcctctca tggtgagaca gagacacaag agactcctct gaagctttca 22920 ggccagcaag cctggcccag aacagaca aacagcaaag accctgcttg aaacacagtg 22980 gaagatgaga ccagcacctt aggctgtcct ctgactcga aatgcacgct gtggtacatg 23040 ggtgcccaca ttcacaca tatagagata aagactggct gggcagtggt vakacacgcc 23100 tttaatgcca gcacccttga ggcagaggca gttagatctc tgagtccaag gccagcctgg 23160 tctacagagt gagtttcaag acagccaggg cacacagag aaatagaaa aggaaaaaa 23220 aaagatata aagactgaa ctgaaaataat atattag ggctagagt atagcttaat 23280 ggcatggtgc tgtctagca tgtatgaagg tcctggttta atccccagct tgaagcatg 23340 tgtgtgtgtt tgttctttt tgtgtactg gggaactgga cacaagcct taggcagtg 23400 ctctgccatt gaactacagc ctcagctttc ttttcttt taaaaatgtt tattatttt 23460 atgtatatga gtactctatc tgcatgtatg actttatgct agaagaggc gtgagatccc 23520 actatagatg gttgtgagct accatgtggg tgctgggaat tgaactcagg acctcaggaa 23580 cagcagccag tgctcttaac cgttgagcca tctctccagc cccctcagct ttctttttac 23640 ccccccacac acaatatctc tctaagttgc ccagtttagc cttgaactta ctctgtagcc 23700 taggcaagct tctaatttgc catcctcctg tctcaatttc ctaggcacct gggagtacaa 23760 ggccatgtat agctttatat atatgttcg tgcagtgcct tcaaagttat ttctcaaatt 23820 agagaactga gttttgactc tagccagcca gtcttaaaaa ggaatgaaaa taaggcctat 23880 tctacaaatg aatgaacctt gaaaacagaa ttcttgtggg gaaagaatca tgatcccatt 23940 tctatgaggt ctctaggata gatcaatgga gcaacagaaa gtagagtaaa ggtgagcagg 24000 gtctcgggag agggctgaga accattattt actgagtaca gcttctgctg cctggtgcaa 24060 aggttctgga aaccaaggca atgcttgcac aacatgatta gttacttcat gctagacact 24120 tcaagcggcc acagttgaaa aatgttgaat gtgcagattt tagcacgggt gttttttctt 24180 gaaggtctca ttgtgtagct ctagctagaa tttggaactt gaaatgtaga ccaggctagc 24240 ctaaaattct caggagatct acctgcctct gcctcctgaa tgctggctgg gattaaggga 24300 gtgagctacc acacctgacc ccttagtaca tttttttttt taaatcacag taacaccact 24360 aagtcactca gctaaccaca gtttgacatc agtttttatt ttctctaagt ttttattttt 24420 gttgttgttt tattttgttt ttcaagacag ggtttctctg tgtaacagcc ctagctaccc 24480 tggaacttgg tctgtaaacc agactggcct tgaactcaca gagactcacc tgcctcagcc 24540 tcccgagtgc tgggaccaaa gatatgactc ctggcctttt tattttcttt ccatgtgtgg 24600 gaggtagggg ttatgcacct gagtgcagtg cccacagtgg tcagaagagg gcaacatatc 24660 tcctagagtt gcagttacat gtggttgtga gctggctgag gttggtgccg ggaactggac 24720 ctaggtcctc gggctgagtg gttccctttt tctgtttttg ttttcaatac agggtctgat 24780 ctggcccagg ctgtccttga actcctgatt ctacacctcc aaagtactgg aattatagtt 24840 acattttcac ttacaaaaat attagctgtg gaaatagctc aggatatgag tatgtgctta 24900 gcatgcgtgc tgccctgggt cctatcccca gcacacaaaa gaaagcacat gactgtcatt 24960 ctttgttagc ccccagtgcc aacccaggct ttgagagagt tcagctttgg gtagacacaa 25020 aggacctctg gttagctagt ctcccaccct gctgtcccaa tctactcact tcacgacgcg 25080 cttggcccgg tagcagtgca ggtcatagga aagtgtgtat gtgtcataga gggtcgcctt 25140 cacgttcagg cagccgatga agtcctgggg gttcagcttg tataggtcaa aggttacccg 25200 ggccacatca atcttctttg ttggcttctg ggaaagggat agctgtggtc tcgtcttgcg 25260 ctgcaagaca agtccatgtt agctcgcctc tgggactcct tccacgccct cgacactcac 25320 ccctgctgga ggagactgtc acctgttctg atgggggctt ccacttctgc cccttctgca 25380 ggaccatgaa cacagtatct cttgccaggg cttggaagta ttcttctgtc tcaacaatcg 25440 tgccgtcttc ctccagcacg agggagaagg gcttgtcttt aagtttcaag atatcctggg 25500 cctggaaaag aaggttggtg acatttctgc catccctatg gagccacaga gttgaggagc 25560 aaaggcccgc agtaaagcac ttcagctgcc agagagagag caagtgagct gccatccatc 25620 cgtgcgatgg actgactgtc actgtggggg cccactcaag ggctgtgtct cagtacttcc 25680 tactcccaag aaggagtgta gtctaataga atgggagtca tatgtgaggt cttttttatt 25740 gagaaataaa ttacatacca tgctgggcaa tattaggagt catatcgaag gccttgggca 25800 agctaggcaa actgcagcat tgaactatat ccccagcctt ctttgtactt tttgtttgga 25860 gacaggatct tactaagttg tttaggttgg cctggaactc actgtgtagt ataggtaggc 25920 ctcagccttc tatttagatc ttcttgactc agcctcctaa gtaacttgga ttacaggcct 25980 acactaccag gcccagctaa actttatttt gttggtggtg gttttttgtt tctttttgcta 26040 tgtagccgtg gctggccttg aactcacaga gatccacctg cctctgtctc ccaagtgctg 26100 gggttaaagg cacgttgtta tggaataatc ttttggtaca ctgtgaagtt gtgtctttgt 26160 caaggcgctt tctgactggt ttaataaaag aactgactgg ccagtagcta ggcaggaggt 26220 ataggcagga aagcaagaca cagaggactc tgtgaagaag ggcagagtct tgggagtcat 26280 gagcaaatgc agagggaagc aagatgaaag aaggtaccac tatgatgcag agggtagata 26340 gtaaaaggat taatttaagt catatgagct agctaaacac aactctaagc tatcagccaa 26400 gcatttataa ctaataatga gtctttgtgt agttatttga gaactggcta tcgggataga 26460 aaagtctgtc tatagtgtgc atcaccatac ctggccacta ttttaaattt ttatttttaa 26520 ttatgcgtat atgtatgtat gtgggtcccc tggagcaagt gattgtgagc tgcccagtgt 26580 gagagctgggg aactgaacct ctgtcctctg aaagtgcttt taacgactga gccaccactc 26640 tagccccaac aactcatttg taatcttgtt cctgccaaag ctacatggca tgaactgaag 26700 aaacaggtca ttcaaaactg agggacagga aataaaataa ctggcttgcc atctttaaaa 26760 gccagctaga agaatcagca ggtaagggta cttgcagtgc aaactttagc atttgagttc 26820 aatacctgga gcccacagta gaaggaga aatgactccc aaatgttgcc ctgtgacctc 26880 catgtgtacc ctgtggcatg ggcatgccag tgctcacaca cacgcttcat agacttacag 26940 taataatttt aaatataaaa taaaactgtc tggatttgca ggacaatcag cagtgctcca 27000 acagagtctc aagataagct tggggggtt cttgttatgt agccctagtt gggcaggaac 27060 ccctatatag agaccaggct gacctcaaac atgtggtcct cttgcttctt catgcatcac 27120 cacactcagc cttagtttta atttcttttg aggcataccc agcgtatcct ggaactcaat 27180 tatgtagccc aggctagcct caaacctgag atcttccata tctggtctcc cagatacgag 27240 ccaccatgcc tggctcattt ctccacgtgt aaatatgggg aaggggaatg aatggccctt 27300 tcagcaagaa aataaccacc cacccacggt tggtgtgaaa gacggtagtg cactgccctc 27360 cttctgtcac ttgaccatgt cacttatcac acacacagct ctccaggcgg aaatgccaac 27420 cctccagtgc caacaccttt ctccgcttaa gaccatgcca ctcatgcgca cagacttcag 27480 ggctctccag aaaccgaagc agccggcagg ctcaactctc agccaaggcc tggagagagc 27540 ctatgcaggg taccggcaga tcccttacct tgcccaggag gtcctccagg ctgtgagcca 27600 tgatgccttt gcgaaccttc cgatctgccg tgctaactcg acagggcctc gccctgggga 27660 cttcccggct gggcttagac accagctgtt gggtcaccac tgcagtgctc actgctacat 27720 gcctggggac aacagtttgt acagcagatg acctgcctgc ggctaccagg tctgcccatg 27780 cctgcctgct tgctgagttc aggtgtgacc tttccttcca accaatcatg gctgcttcag 27840 gcggctttca caaagtccat cagaagacac agctccgatt tggaggggca gggaaatcag 27900 cgggaatctt gcttaaatca gactcacttc agtgcctctg agtggagccc aaggactgct 27960 aacaagtccc caagctgtgt cagtgtggct ggttcttgga ccagccacat tctcctgtgg 28020 atggcttctg cttttgtggc cttgttcacc ttgcctaaga ggcagggtga aatggagctg 28080 gtgtgtgtat atatgtgggg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 28140 tgtgtgtgtg tgtgtaaatc ggcctgtaga atatgtgggg actctgaggt acacagggta 28200 gaaagaaagg agcagagaga atttgggtct tggctggaat tccaagtttc tctctgttac 28260 ggatttgcta ggtgacagca tacccagcct gtgactcagt ttcctcatct gttgacttgg 28320 catagttgcc accacttccc agagaggagt gaggctcctg ggagatcagg aacaaaggca 28380 cctggtgccc agcctgaccc tttctactcc agatgtgatg ggaactgtgg cagtgacaga 28440 gagtaagatg gggactgtct agactgcaag atcttgggtg ctaccctagg cctcctgaat 28500 cagatctgtg ggggtttatt gtaagatttt gtctgttttg ttttaatggg ggtcttgctt 28560 atttgtttgt tttttgggat agcatctcca tatatagccc tggtttactg gaactaattt 28620 cctcatctct gtctctgtct ctgtgtctgt gtctgtctct ctctctctct ctggacctgg 28680 acctatgcag accagactca aactcaccaa gatcctcatg ccactgctcc ccaagagcca 28740 ggattcaaaa catatgccac catgcctggc catatcagct agaccccaca tgactttttt 28800 tgtcttttaa aatgggatgc ctatgtagcc ctgactgacc tgggacttgc tgtgcccact 28860 acaggctggc ctcaaactca gatctgcctg cgtctgcctc ctgagtgctg ggattgaaag 28920 cgtgtaccat cacatccagc ccctacatgg tttttatacc tattaaagtt ttcaaagttc 28980 taatagatgg ctccccagct ttccccttcc ttcccatacc cagctctcct tacctggaca 29040 gtgacttagg gtacaggagg ctgagagact tcatggcata gtccatcctt gtcagctgga 29100 ttgtgttgga cctggatcgg aggcagtaac ctatctggtt agagtgggaa gggagggagg 29160 ggtccctctc tttctttctt tgactcagtg gatcccagcc cccacacggc aaccctctgc 29220 cccagtctct cctcctaccc ccaatttccc tgaatccaca ccaactacac tgagtcagcg 29280 tctaccatcc ccacccattc cacagcccca gaacaactca ctccatgttt ctctgcccca 29340 agctctggtt acactgagct gaggaaccca agtcaaggct gaagaggccc tcactgattt 29400 gtccctgtcc ctgggggcag agtccacatt atgctggcag gaggtgagtc ataccacctc 29460 atgctgtgag agacttgaaa gtcacctatt gtccagaaac tccacaggca gtcagacctt 29520 ggagagcctg ctctaaccat gggcccttga gcaataaagt cctcctgagg cttagaactg 29580 gctaagagaa ctttctgtga tcttaaagac tgtctaggct tgtgttcacc aataccttag 29640 ctgttaccac tgagcccttg gcaccataac taatgtgaca aaggagctga caccgaactt 29700 ctattgaggc ttaactaatt tagttaggta actactgtgg tttgtgacgc caggtacccc 29760 actggacatt acagttgtag tggagtaatc ctgggctcat gtgccccctg cctttcctcc 29820 tatattgctg ttctctggtt cttgcctgct gagctgcaag tctctgcaca ggcagtctct 29880 tctacctgca atgcttgtct caaactcaga gctcaacata agtcacctag aagccccagc 29940 tcctggctgc caactcagga aagccttttc tggtgccctc ttgcctggta ccagattgat 30000 ccctgtgccc tgcccttcca tgttcatttc tgctgcagct ctcgggtccc cactgtgcct 30060 gttttcagcc cgtagcacag ggacccagtg tgaacaccag gagtgggcag gcatggagaa 30120 ctgcctcctc agtgaaggaa aaccattctt cccttctgat aactcgggtt ctccccggtc 30180 aaccttacag agttagagac cctcgtcccc acttaatgct acccaagcac tcttgtttca 30240 ccctgtttct aatacccacc actcacacag ctccacaaaa cccaccacac ctgctctgtg 30300 gtagccaaat accccagctc ttctctccc cctcacctcc tagcctcctg gcaatactgc 30360 gtggccaggc acagcccagg cctctcccat tttatttcta tctcctgtga agccactccc 30420 ttggccccag ccccagctgg ggaaaagagc acagggagtg agtctgaaac tctccctagg 30480 gatttcaagc cagactgact tccagaaagc cccgaaaggg ggaagaaggc atgtttcgaa 30540 agctctagaa ggaagaggta ctgcccagga cttggtacca ggcaagaggg cacagaaagc 30600 aacagacggg gcttgtggtc tgcgttccac ccctcacacc agctgtttgc gctcacccga 30660 gttgccttct ttcagccctg gattttctcc tgtgcatgag gaataacgct cctgttgaag 30720 ccaggaatgg tggctcagag ctgtaatctc agcacttggt agactgaggc ataaaaaggg 30780 ccatgagttt gaagctagcc tgggctatag agttaggtcc tgtctcaaaa acaaaaatat 30840 agtcagacgg tggtggcaca cacttttgat cccagcactc ggaaggcaga ggcaggcgga 30900 tctctgtgag ttcaaggcca gcctggtctc cagagcgagt gccaggatag gctccaaagc 30960 tacacagaga aaccctgtct cgaaaaaaca aaataaataa ataaataaaa aagaaatatc 31020 tttttttttt tgagtcaggc aagatacttg agttttatta tgtctgtcta aaagcaggtg 31080 ttaggcctat ttgtagtgaa gcgtggtgtg ctggcgctgc aacacccggt gaggaagcag 31140 gaacctgatc tcagagccca agaactgctt gacagcaggc cttcggcact tgccctctga 31200 aatctcttcc accttcatca ggttaatgga gtgtgcacgg gcacagtgcg ggcaccccgtg 31260 tcttggtagc actgtttgac ggctccagca gcggtcaggt cccggtattc ttggcacatg 31320 ttgtgtgtgt catgttgtga gttgtagtgc agccataccg caaagttctt cacccgcagt 31380 ggggcctcag gtcgcactgc acagtgtacg atctcctcgg atgacttctt catcttgttc 31440 atctgtgccc aaaatcggga cttggccacc acatggttcg tcgcaaagat gtgcatgagg 31500 tataggagtg gtgtgtggca cttccgggtg ggcaagcagc gccccaccac cttgtactcc 31560 cgaagcgtgc ctgaggcctt cgtggctgat ctgtccttgt tggccgccag ccacaaaaaat 31620 aaaagggaat atcttgccta taatgttaca atcccatcca gcacttggga agccaaggca 31680 ggaaggcctc tgtggatgag accaggcctgg gctacagagt gacaacctgc caagagaatg 31740 gggtgggtaa tcccaaatct tgggtatagt gaagtacagg actcaagaga cagaggcagg 31800 aggatcatct ccagttcaag acaagcccga gctataaggc ctcaaaaaca aaaaaagaat 31860 attagatcta tgatttattg ctataaacac tacaacttaa aaaaaattaa aataaggaca 31920 gcaagatgac tcagtgaatg agggtttgaa tttgatcccc agtacccaca tagtacaaag 31980 agagaactga ctcccatagg ttgtctttcc gtgaaaactc tccctccctt cctccctctc 32040 tctttctctc tctctaaatg aaaatgttta catcaaagcc tctacaaaga gcattcatgt 32100 tacacctagc gttggagaga tgtctcagtg gtcaagagcg ctgacttctc ttccagagga 32160 tccaggttct attacaagta cccatgtggc aattcacaat catttataac tcagttccag 32220 gggatctgac tcccttca ggcctctgtg ggccttcatg tgtgtggtgc aaagacttgc 32280 acgaaggcaa aacactgtac acataaaata aaaataagag acagaaaagg gaatttattc 32340 agtgtggcca taccagaaaa gatgagagga ccagtctctt caaccctgtt tttggagtgc 32400 agatggtagc tctaggaatt tacaggagaa gaacaaaaaa tgggatgagc tggtggtgca 32460 ggcctctcag tgcttgagag gcagaggcag aggcagggga atctctgtgg gtacaaggac 32520 agcctgactg acctggtgga ttccgggcta accgaaacta caaaataaga cactacctct 32580 aaagttaaaa caaaaaaggg aggcaataag tatgcatagc taagcattct attctattct 32640 gcccaaacca ctctaattga cggctacctc cagtctgaag taagggtcct gcagtagcta 32700 ggaagaggac tacctcccag gctgactgtt cctggctctg gcaaaact gaaaaggaaa 32760 actggttcag aaggagatca gagcagaggg gccaaattag aacgaggatt gtgccttcct 32820 tcaggtttag aacaaccact ggagaatttt aggtactgct gtggtagttt ggaagaac 32880 gaggcaaaaa cttattactg gcaatctgta atgtccccat aaatccttt tatatctatt 32940 tttaaaaaga tttctatatt atatgtacag cattctgtct gcatatatgc ctgcaggcca 33000 gaagagagct ccagatttta ttacagatcg ttgtgagcca ccatatggtt gctgggaatt 33060 aaactcagga cctctggaag agcagccagt gctcttaacc tctgagccat ctctccagcc 33120 ctctatttta ttttttaaaa gaattatttt atgtgtatga atatttgcat gtatgtatgt 33180 gcaccacatt catgcagtgc ctatggaagc cagaagga cgacagattc cctgaaacca 33240 gagttacagg gggctgtgag ttgctttatg gtgctgagaa ctgagcttcc ttcctgtaca 33300 agaaaagcac atgctgttaa ccactgagct gtccctccag ccctccccac ccctttgtgt 33360 gtgtgtgtac ttgtatgtgt atgtatgtgg acatgtgagc atgtgtggaa gtcagaggat 33420 ggtttgcagg agttggttct ctccttctac cctgtgggtc ctagggataa aactcaggct 33480 ggtagcatgt ccctctacct gcaaagccat ctcctgggct ttgtcccaat aaatttcttg 33540 ctctcccttg gtatcttcat tcttaaaggg aaggacagag ctacaggtac aatgggtgaa 33600 aattggcctt tagttaccag tcttgaagat gagtacttaa atttttttca gaatcctttt 33660 ttttttttta aatcttttta tttatgtata cagtattttg tctgcatgta tgcctccagg 33720 ccagaagagg gcaccagatc tcataatgga tggttgtgag ccaccatgtg gttgttggga 33780 attgaactca ggacctctgg aagagcagac agtgctctta acctctgagc catctctcca 33840 gctcaagaat cctttttttt tttttttttt tttaaggcag ggtttctttg tgtagcctta 33900 gttgttctgg aattaggttt gtagaccagg ctggcctgaa ttcaaagacc cacctgactc 33960 tgcctcccag ggtgctggga ctaaaggtgt gtgccacgac acctggctaa gatgagtgct 34020 ttttaaatgc ctatcacatc tacaggcaca actgattggg aagctgattc aaaccttgag 34080 atagcaaagg gatagaggtg ttgatgcaac ataaagacag agatgcccag attttctcct 34140 tcctttagtc actgggcggg actctgcagg tccagtgagg actcactggc tctcttgtta 34200 aagccttatt tagagttaag ccaatccttg gaagaggcat cattcattca ttgaacactt 34260 gcagtgtgtt accctgttaa ttcagcagaa agcaagacag ctttaggcca cacctggagc 34320 agctggacca gagaccaagt ggaagcagga atgctgatgt acacttctca tcctgatccc 34380 ctggaggctg tcagcctggg ccacacagtg cgagatcctg cctcaaaaac gtttttgcta 34440 cccattggtg attgtacatg tactttgttc tagcactcag gaggtagggc cagcctggtc 34500 tacaaaggac agccaatgct acacagagaa accctgtcta gaaaaacaaa acaacaacag 34560 aaactttttt gtctgatgtt tcatcttgtt tcttttctaa taatgtactg ggcagagaaa 34620 aacaaaataa tgcagttggg cctggaggct tagacctggg attccagcac ttaagagata 34680 gaggcaagct cattgctttg agttaaggt ccctctgccc tgcatagtga gttgcaggtc 34740 agtgtggact atagagtaag accttgactt tcaaaaagca caaggggcca ggcgttggtg 34800 gctcacagct ttaatcccag cactcgggag gcagaggcag gaggatctct gtgagtttga 34860 ggccagcctg gtctacagag ggagttccag gacagcctcc aaaacaatac agagaaaccc 34920 tgtctcaaaa acaaaacaaa acaaaacaaa acaaaaatgt caaaaagcac aagggccatc 34980 aaaatggctc agtgggcaat tacaaacttg atgacctgag tccattcctt gagacctaca 35040 taatggaaag aactgactcc cctaagttgt cttctgacct tcacacatac cgtgtgtgtg 35100 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tacatgcacg cgctctcgtg 35160 agcaagtacg ctcctgatac taaattaata aaaaggatta aaaaagtaaa accacaaaca 35220 tgaaccaaac caatgtggtg tataaccta tgtctggaga actaacaggt ttctggaccc 35280 agagaggtag agaagataat ctaatgtctt caggggaatt agggggataa agcatcagag 35340 cataagaaa gttccctca ctgggaagt agtggaggca ggagcatcag gattcagg 35400 tcatctcatc tatatagtaa gtaagttca gactagcctg ggttacatga gaccctgtct 35460 aaaaaaaaaaaaaaaaaaaaaaaagg gagcatggt gcacagatt 35520 tgagattgaa acacaaaaat agctcaactc tggggcagac agcagagtca ggggaaagtc 35580 taaggaagtt ctgggaagg gtcaggaggc ccctccctgc ttcattcacg ccccaacat 35640 ctattaaaca ttccatgtgt acttggtgtt tctggcctga ggatgactca cagaggctga 35700 cacactaagt taatcacagg aagcacagaaaaaaaaaaaaacaggcct 35760 aatgcaatgg aatgcctgac tgaggggtt cttcacagtg ctttggac gaccctttgt 35820 gtctcattga attcattc tcagaaaca ctcctcaaat gtttacacat tgtcaggcac 35880 ttgagctgc agggacacag aggactcagt tctgtccct gcctttgga ttcacaattg 35940 ttgctctcc aaagaactgg ggttcggctc tcagcaccca cttaaggagg cccatgacca 36000 tttgtaactt cagctcaacc gcttccacaa gtacccacac acatgtctgt cccccaaaca 36060 cacctatatt cttcatgcta accttgtgac actatgagca aaatcaaaga cattaagaag 36120 ccaggttcag tgccctgtgc cttagtccta acatttgaga ggctaaatca aacaggaatt 36180 gaggccactt ggacagcatc aagagaaatt atctttgaac atatgaaatg atattatcac 36240 agaaatagaa ttgcaattta aaccaggtat gtgatcccct ctccccccac caaaagaaaa 36300 aaaatagag ggctggagaa atggctcagt ggtaaagagc acctgctgtt cttgcaaagg 36360 atctagattg ttttgttttg ttgagacaga gccacactat gtaactctgg ctgtcctgga 36420 attcactatg tagatcaggc tagctggtat gtatgtctgt ccacaaatgc atgcctggtg 36480 ttagaggctt ccagaagtgt gtgtcagacc ctctggaact ggagttatag gtggttgtga 36540 gctgccatgt gggtgatgta attgtacttg ggccctttgc aagagcaacc agtgccctta 36600 acccctgata cttgctttta aggaatttgc atttttgttt tgaaattaca tgttggaaaa 36660 gttttccaca tcagtaagaa atgccatcct tactatttct tcctgcagct tctcagaaat 36720 atttttaat ctttttttt aagattttat ttatttattt attatgtaca caacattctg 36780 cttcatgtat atctgcacac cagaagaggg caccagatct cattcaaggt ggttgtgagc 36840 cactatgtgg ttgctgggaa ttgaactcag gacctctaga agagtagtca gtgctcttaa 36900 cctctgagct atctctccag cccctcagaa atattttaat tcaaaaatct ttcttagcca 36960 gctctcaagc cactggatta cttcttgctt ctgctactga catagacttc atcttgattc 37020 actccataca gcaactagat gtgtgtatat aaatgacaga tagatcatag atggatggat 37080 ggatggatag atagatagat agatgataga tagatagata atctcacttg ctacctaggc 37140 tgacctcaaa ctcatgcctc agttcctaa gtgatgtgat tacgggcata cactattatg 37200 cctagcataa ccatttgttt gccttaaaat tttttaagat taatttttta ttatgtatac 37260 agtattgtgc ctgcaggcca gaagagggaa tcagatctca ttacagatgg ttgtcagcca 37320 ccatgtggtt gctggaactt gaactcagga cctctggaag aacagccagt gcttttagcc 37380 tctgagccat ctctccagcc cccagccttt aaaattttta attaattttt tttttgtttg 37440 tgtatgggtg tttggcctgc atgtaggcct atgcactata tgtgtgtagt actcacctag 37500 accagaagag ggtgccagag actctggaat tggagtttca gtgcatgatg agctgccaag 37560 tgggccttaa tccccaaacc ccagagggtt gaagagggat cagtggctaa gggtgcttgt 37620 tcttgcagag gacccagttt tgattcccag agcccacatg gtagctcaga acaagaactc 37680 cggcttcaga ggattctgca ccctctctgg gcctcatctg gtaccaggca tacatctggt 37740 atgcagatat atacacgccc tgagtggtga caaacactct gtgagttcaa ggctggtcta 37800 catagagaat tccaggacat agtgagaccc tgtctcaacc aaacaacctg gatcctttgc 37860 aagaagagca ggtgctttta aacactgggc catctctcca gcaccacctc ccccctttaa 37920 tgatacatag gtcccacata gcctagttcg gcctcttaac tcctgacata acacctcatg 37980 aattcctcat cctcctgcct ctaccttttg agtgagtgac gagattacaa acgttcgcca 38040 ccatctttgt tcccagttgg ccctcaaggc cccagggtct cagttcagat cctgccacta 38100 tggatacata gcattgaaat ggggaaggct gcctctgtgc tgcctcagcc tttgggtgac 38160 gcctcttgct tggcactgca ttttcaggag catgacatct ttgctctgga ccctgacccc 38220 aaggttgtgt ggagctgtaa gaagtgtaag ggtgtctggt attggtggcg cgcgccttta 38280 atcccagcac tcgggaggca gaggcagcag aggatctctg tgagttcaag gccagactag 38340 actacagagc gagttcagga cagccagggc tgttaacaca gagaaacact gtcttgaaaa 38400 gcaaaacaaa ataaaaccaa caaaacaaca acaacaacaa agtgttaagc gcgcatgcaa 38460 ccagccactg ggcaattagt caaagatgcc aagtctagat agacatgcag ttaagaactt 38520 agggtctaga gagatggctc ggtggttaag agaccagaag agtttgcatc ccagcgctcg 38580 cacagtagct aacaacagtc tatcttacac cctcttccag cctctcctgg cacaaggaac 38640 acacgtggtg ctcatagtta caaaacagac acaacacgca tacacaaaga aataactaat 38700 ttttaaaatg tcttgtaatc aagtaaaagt gctaactcta gggactaaat taattcctca 38760 gtcgccctac tccaggagcg gtaaggctgg ccagaaagac caagaacgca cgcgcggagg 38820 agaaaccaca gagtcggtcc tcccgggata gagaggccgg aagtgctcgc ggagctgcac 38880 gccgggtgct ggaagcctac tgagccccga ggaagggctc cgctcggggc ttggcgtggt 38940 gggtgagccg gagggtcggc gtgagcggcc tgggctttgg ttctgaatga tggcgtctcg 39000 ggcaggcccg cgagcggccg gcaccgacgg cagcgacttt cagcaccggg agcgcgtcgc 39060 catgcactac cagatgaggt atgaggtgag ccaggagcac tgaggccttc cccgggagga 39120 gcctgcgggt ctcgggaagc gacgcgggcg agcctcacgg tgccgctccc ccagccagct 39180 gtcgcgtact accgggtccc cggctccggc gagcgcctcg ggtctgttta caggccggga 39240 agcccagtgg cctgccctcg cccgcctcgt gctttgaggg gatctggcct gcagaggctc 39300 aggggtcgat gctcagcccc tctgaatgac cttggagaca tcatttttct tttttcaaat 39360 cgaggtcccg cagtgtctag agttcaggct ggcctggaac tcacggcctt ccctcctcag 39420 cctcccgagt atgcgcctgg tctgaaaact aacattctta aagaaaactg cgtgtgtgcg 39480 tgtgtgtgtg tgtgtgtgtg tgtgtgtgcg cgcgcgcgcg cgcgtgtgtg tgtttgtgtg 39540 tgtgtaagtt tgcccacacg agagcaggtg tgctggcaga atgcagtgcc agtaaagggt 39600 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 39660 gtgtgtgtgt gtgtgtgtgt gcgcgcgcgc gtgtgtgtgt ttgtgtgtaa gtttgcccac 39720 attcttaaag aaaactgcgt gtgtatgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 39780 gtgtttgtgt gtgtgtaagt ttgcccacac gagagcaggt gtgctggcag aatgcagtgc 39840 footaaggg tgttggatct cttggagcta tagttacaga cgtgtttaaa tccacctgag 39900 actagtactg aggaccaaac cccgaagtga ttttgtgaga tagcggcttg gtatggggtc 39960 catgctagcc tccaggagcg gcaggtgctc ttaaccactg agccatctct ccagtcccca 40020 ggctgtcttg gaacttgctc tgtagaccag actagaaatc agagatctgc ctgcctctgc 40080 tgccaagtgc area ggtgtacact acagccgccc tgttgaccct gtctttagga 40140 agatgtcatg tgtactttac aaagagtttt gatgtagtca tgtcttaagc tttgttgtca 40200 cacaagatga aattaattac caccaggggaa tttttcatca aattgtgctg tacttaaata 40260 tgcctaaagt aaatacttg gggtcagact cttgaagtttt aaaccaacac caggtactga 40320 gttgtgttga acccacacaa ctaccttgcc tcctgcagat tgttaactct gctggctgtg 40380 gtggttgaag agacccccac attggcttag ctgttaagag cacttgctgt tcttggagag 40440 ggtctaaact cagttcccag caccgacatc aggaggctca caaccaaaca cctgcaactc 40500 cggttccagg ggttccgatg ccctggct tcccgacagc acaggcgtgt gcgtggtgca 40560 cacacataca catgagataa atttttaaaa agaaagaaaa gccaatctgg gtcctgcctt 40620 caaggagctt gagtgccaga agtgattctt ttctccttac ctcccacatt tcctataaca 40680 aattaattt ccccttaaat ccactgaatt attcaactgg ttcttggcct cttctttac 40740 agaaggatga caagcccttt ggacccttct ttttcaactt taggcccctg cctaatggat 40800 gctcttgttt tcatgcgtac ctagagcaga caccttgctt gtgacaatat gaaaaggata 40860 taattgagac cccgagaaga tcaggactaa gagatgtgcc tccttctcat ggaaactata 40920 tctggagaaa tacagggagc ccaaactgtg ttccttgttg ctaggcctcc ccctaatggc 40980 ctttgtttct gtgatacgaa gccatctttc taaccacagc tcagaattct tgtcatggct 41040 tggtgagata atgtcttagt acgttgtcca ggccaacctc caatcagaga tcctctgcct 41100 cagcctccca ggtgccatta ctgttcttca gtcttgaagt ctacacatgc atggtctttg 41160 taactgttac tacctctctg tgccttggca gttgatttcc tgaaacacaa atactaaaaa 41220 gcttattcct ttgtgcctgc atgtttttgc ctctgcgtag aagcccactt ccactcgtct 41280 cattacctct gctgaaatag ctctctcctc tagatataac cttctctggc tttccatctc 41340 atgctaattg gttcttccct gggatagcgg atatcttcgg tttatatgta tttacttgag 41400 ttttatactt catgtttccc agcctctatc agaggctgtg tcattcagat tgtatctatg 41460 tgtccagtac cggtgcctgg tggtcggagc aatttaaatg aaaattgctt ctccctctgc 41520 tctgccttac agtgtgacgc tcaagagtga aatcaagaag ctgatctacg tacatctggt 41580 catatggctg ctgttggttg ccaagatgtg tgtgggacac ctgaggctct tgtcacatga 41640 ccaagtggct atgccctatc agtgggaata tccatattta ttgagcattg tgccctctct 41700 cttgggcctt ctctccttcc ctcgaaacaa cattagctac ctggtgctct ccatgatcag 41760 catggggctc ttctccatcg ctcccctcat ttatggcagc atggagatgt tccctgccgc 41820 ccagcaactc taccgccatg gcaaggccta ccgcttcctg tttggtttct ccgctgtctc 41880 cgtcatgtac ctagtgttga tactggcagt ccaagttcat gcctggcaac tgtactacag 41940 taagaaactc ttagactctt ggttcaccag cacacaggag aagaaacgta aatgaagcct 42000 gcctgatgga cacatgaagg gagctgttca gaatctccat ggactgtggc atctgtgatg 42060 ttggcaccta gtgcacacta tcctcagatt ttggccttga gttctctgtt accatctgct 42120 gagatgacaa atctgtagtg tttaatttat tcttgactag ccacaaaccg gatgaccgat 42180 gtctgtggaa cacttcaagt tgaggccctc cagactgagc cttatccttg ccttctcttg 42240 gtcaaattcc ttacttccat ttatcacctc ttcatcaccg atactaaaag agatctggaa 42300 taaatcagtg cagaaattct acttcaatct gtaggtcatg gggcaagcaa catttgggaa 42360 gttgctcccc taaaggctac tctgtttact gcaaacccatt ttaattaaaa aagaacttca 42420 ataaagttaa gactgtcctg tgctttgtgt ttgagatttg attgaatttc aaagttgtat 42480 tgctctagag gacttagaca ttatgagaag aatagatgtt tcctgagaac atgggactgg 42540 cgctggagga gagacagtgt gaagagtctt aagtcagctg cagccctcta gctcttaggt 42600 gagaacaacc ctgggatggg ggttgctgga ggaatggctc aggggttaag agcactgaca 42660 gaggacacag gtttagttct cagcacccaa atggtgactt aaaacccattt gtaactactg 42720 tcccagggca tttgacgcct tcttgcctcc ggatgcacta ggcagacatg aggtacaaaa 42780 acaaacatgc aagcaaaaca ctcaaaaaat taaaaaaaa aaacatggtc tctcaatgta 42840 gctgtggatg tactagaact cactaagtag agcaatctgg gctcaaactc acagagatcc 42900 tcctgccttt gcctaccaag tacagggatt taaggtatgt gccaccacac ttagcaatat 42960 atatttggcc agacatttaa tctccaccca taaagcagct tatttggctt ggattatgag 43020 ctcccctttt tatgcttgtt tgagacaggg cttctctata gctctggctg tcctggaact 43080 tgcttcgtgg accagactgg catcaaactc aaatccacct gcctctgcct cccacgtgct 43140 ggcattaaag gtgtgtgcat cactgcccag ctgggttacg agctttctat ttggttattt 43200 atgtttatta gacttttatg tgtctaaaac tgagtaataa tacccaaaaa gaatctgctt 43260 gttggggcta taattttcca gtaactgaca aataaacctg agtcaaaata agaagaaagg 43320 ggtttgagtt ctttttccct tttagtttct gtcattgatt attccttggc ctgcttgttt 43380 ggggaatgtg caaaggaagc tgctaactct atggaagcca gaaaaagaaa aaggtgagat 43440 tccagtgtcg ccactagggg cagactgcca gtgacgtaac ttcttcactc agcccctccc 43500 aaagcttcct ccacctccaa aaatgtcaag gactggtaac tagtacatgg gcctttgaga 43560 aaagatagca gggagcttcc agtcttgggg gaatgtgcag tgaacataac atctaattgt 43620 gtgagagacc cacagctgag atacagttta agagtcacac ctagaagtct tcacagtaaa 43680 taaggcagca aaggtgtgca tccctttgca ctggacttta ttatgcctag atgatgctca 43740 gatctgtaac tgcagagatc tggccactac agttgtaatt cttttgtcct ggaaatgtgt 43800 tgcttgtaca gtctgcaaag ctaattcctc cagctatccc actgatcatt tctttggagc 43860 aggggcagtt tccagacaga attctctgt gtagtcctcg ctatcctgga actcagagat 43920 ctgcctgctg tgtgctggga ctaaaggcat gtaccaccat gcccagctcc tactgatcat 43980 ttcaaagata acttcttgtg cctcaggcct attgctgttc ctgcttcctg tgtttacggg 44040 aacacagctc tggatcatcc accctgcaag tttaagtccg tccccactcc atatacacac 44100 tgcctattcg gtgaacctcc tatagatacc ctgataagtt ccctagcttc tcagtttaac 44160 acttagaact ctccctactt aaggggaggc ctctcagtgg tgaagtgctc ctctagcatg 44220 caccgaggcc tgggtgtgac tccctcgtaa caacaaataa aaacaaacct tttctgctct 44280 tacattcctt tattaatctt taagatgcca cttgtatgaa aattcctcaa ttttcaattc 44340 ctcaatttga agaacaactt taatttgtct ggttgctttc accttattcc tttattatta 44400 ttttctaaca agacagagcc cagaatgacc ttaagctatg tggctgagga tgactttgaa 44460 ctcctgatgc tttgtctcta cttcctaagt gctaggatta caagtatgca ccatcactcc 44520 tggcttatat tactcgtccc ccacccccac cccatagacc aagccttgac ctttattttt 44580 aaaagctgca tatttattct gctttctgac ttagtgtttg tgccttcaac actttcacaa 44640 cccttttctc ttcctcaata aggaaagcct gcttgatcct gtcacggaca cacttggcac 44700 acaaggagcc accacagccc ctgctgacgt gcttctttgt ctagacagtc tcataggac 44760 tttgggtctc acagcatgaa ccccttcaag tctgcctggg cacacaccac atggggattc 44820 aggtgctttc ccagtcttct tggtataaag gtaaacaatc ctgttgccag gggttcgaga 44880 cagcctagtt tcgttagagg ctgtattgta ggaaagccta caatggtatg tcaaacactg 44940 gaccattcga gtgcctctaa gcaccatccc tggaagagga agagctgtat tatttgtttt 45000 atattgacag aattgctact ttgtatgtct tctgtagctc attagtgttc actctgagct 45060 tattgaatga attaacttgc ccttttgagg acaaaggtct gtatttcaca gaaaacagaa 45120 ctggactgaa gcagaaagaa gcccatggac aagggagttt gtcatgagct tagctgaggc 45180 acacattgct aacttcaaac aactgtgatg caaacaatta aaactgcagt gcagaccccc 45240 ctgctttctc ccaataaatt ttccacataa gttctgtccc ccccccaaaa aaaaaacctg 45300 ttagcacatt attagtcaaa gagctaaaac ctgtttttgg tcatcagaat agtgttttac 45360 tgaaagtctt tgaatacctt gtagactaat ttcactcatt gtgaaattag tcaacagttt 45420 taaaacacta tcatgccaac atcagttttt gttagttttg tgcttggaat tctgcttttc 45480 tagccttttc ctataacttt tttctgcctc atggttatgg tgttcacaag tcctcttgat 45540 ttagtgggaa gaatgctatt tgggttagaa agttcagctg ggtatggggg tacatgtcta 45600 atcccaacct ttggcaggtt gaggcagcag gaatactctg agtttgaggc cagcctgaca 45660 man catacata catacata catacata caaacact atctcaaaaa 45720 acaaagttgg agttaaagta tcaaccacag gactaagatc aacctgcctg ttcacaggtt 45780 gctttaaagc tccaatagcc ggttggagag atggctcaga ggttaagagc actggctgct 45840 cttccagagg tcctgagttc aattcccagc aaccacatag tggctcacag ccatccatta 45900 tgaggtcagg tgccctcttc tggtgtgcag atgtacatgg aagcagaatg ttgttacata 45960 ataaatacat aaaatcttaa aaaaaaaaaa ctccaatagc caaagtatta tgaaggcatt 46020 ctaaaattt taatttattt tcattttatt tttggagaca cagtttctct gtgtaaaaac 46080 cccaactgtc ctggaactca ctctagacca gactggcctc caactcacag agataaacct 46140 acctctgcct cccaagtact gggattaggc cgggcagtgg tatcgcccac cttaatccc 46200 aactcgag aggcagaggt tggtggatct ctgtgagttt gagaccagcc tggtctgcaa 46260 gagctagttc taggacagcc tccaaagcca cagagaaacc ctgtcttgaa aaaaacaaaa 46320 aacaaacaaa aagcaagtta atctggaatt aaaagtgtat gccaccacac ctgatgtaaa 46380 tttaaaattt tttttaaatt aatttttcta tacacaggtg ttttgccagc atgtatgttt 46440 gtatagcaca tgtgaacctg gtgcctaaaa agccaaaaga gtgcatctga tcccctggga 46500 ctggagttac aaatggagtg ctgccatgtg ggtgctgaga attaaaacca aatcttctag 46560 aagagcagcc agtgagtgcc cttaactgct gcagcatctt tctggctctt gtgaaggcat 46620 tttatggagt ctgtaacacc atatgggtat caaaagccgt gtggtcacct gatctttgca 46680 gcaagacagg agagagaagg tgtttgagag acttgaggga tggtctagac accaaagaaa 46740 ggtttgtgtg ttgtgagcaa tgactgatta ttaggttgag attctagcca gggagtttac 46800 tatctcttgt ttgatttatt acattttatt gccatcctag gaattaaata gggttttaca 46860 tttgctaagc aagtgttctc tcaacatcct tatatcccct ccccgatttc tgacacttac 46920 tatagctcag cttgtccttg aactcagcaa tcctgcctcc atagtgccgg gattatggat 46980 gtttccactc ccagctgagc ctgttcttca tgtgttgcat gggcattgca gatctctccc 47040 attgctgtga ggctgaaata aatgtggatg aactttgtag tacatagtcg tttacatatc aagatgcctt tccttggtaa acagtgttat gcttgcctct caaattgggga gtgtcttcct gttttaagaa taatcctctt ccctctttttc ttcctctctc ctccttccc ctccttctga cagggtctct ttatagaata ttcctgcctc tctctgcctt ccaaatgttg tgtaggtgta catcaccatg cctgcctagt tagaatgacg tctcagaatg ctggaggtta atttcacatt cttagtccac attatcctga ctggtatgtt caaggtgccc tgacagtagg cagagaccca gcagacctca ggaagtgacc attack gatcctgtag ctgctactca tagcctgttg ggagcccgtg cataggaag aatggcaaga aaaaatggct gccagagggc gtccccatag 47520. fatherctctgt cactagcat gcatgcttca gagcttgcca aaacctcctg agtccctggc ttggtcccca aataaaggtc attagagcat gaagtcttgg tcaactgatt gagactcctt tggagagtgc aaggcttcta tgtagtgcc cctgttgcct cctattctgt ctaatttctt 47700. actctccctg ttgacagctg ccaatctcct ttctcctagg actgtgccca ctcatcactc 47760 actgtgataa gacccctttc cttttctttg ttccttcctg gtctgcctgc ctgctccccc 47820 ccccacctct cccttttcat ggtctcatga aaactgctct cagactcact atgtagctaa 47880 ggatgactgg agctcccaat cctcctgccc ctacttccct aattctggga ttacaagttt 47940 atgccaccac attccacttt ctgatttcct tccttacatc ttaattccac tagctttatc 48000 acactgctaa agctaaaact ttctttgcaa aatgagatcc agaaacccac agactctcca 48060 tagtaggttc tgacagggaa acagctgaca ttgtatggtg ttctgccttc ccatttcttt 48120 gctgtgtgtg tttttggaca agtctcggtc ttcctgtatt tccttccttc cttccttcct 48180 tccttccttc tttttccatt ttttatttga attataaaca cgattgtttt acatgttaat 48240 cccagttccc tctccctccc ctcctcccct accaccatcc ccaactaaaa ccctacctat 48300 cacatatcct ttctgctccc cagggagggt gaggccttcc ataggggtcc tcagggtccg 48360 tcatatcctt tgggataggg cctaggccca cctccgtgta tcttggctca gggagtatcc 48420 ctctatgtgg aatgggctcc caaagtccac acctatgcta aggataagta ctgctctact 48480 acaagaggct ccatggattt ctgaggtctc ctcactaaca cccacattca ggggtctgga 48540 tcagttccat gctggtttcc cagctatcag tctggggacc aagagctccc tgttgttcag 48600 gtcagctgtt tctgtgggtt tcaccagcct ggtctggacc cctttgctct tcattcatcc 48660 ttctctgcaa ctgtattcca gttcagttta gtgtttagct gtgggtgtct gcttctactt 48720 cttccagctg ctggatgaag gctataggat ggcatataag tcagtcatca atgtcattat 48780 caggggaggg catttaaagc agcctctcct ctgttgctta gattgttagt tggtgtcatc 48840 tttgtagctc tccaggcatt tccctagtgc ctgatttctc tgtaaaccta aaattttccc 48900 tctattatgg tatctcttat cttgttttct tctattcttc ccccaactca acctttctgc 48960 tccctcatat actcatcttc ccttctcatt ctcctagctc cttcctcccc ttcccaattt 49020 gctcaggaga tctggtccct ttccccttct ccaggggacc atgtatgtct ctcttagagt 49080 cctccttgtt acctagcttc tctggctttt tttttttttt taagatttat ttattatgtg 49140 tacagtgttc tgcctgcagg tcagaagagg gcaccagatc ccattacaga tggttgtgag 49200 ccaccatgtg gttgctggga attgaactcc ggacctttgg aagagcagtc agtgctctta 49260 accgctgagc catctctcca gccctttcta aaactggaca ttagcctagc ctcaagggtt 49320 gtgatgctaa caatacaagc taaagagagc aatgggcatg atccaaagcc agatagttca 49380 gcaaatattg atttcatccc ttccctttc ggaacagact ccagccacac caaatatgta 49440 aaccagcaag acaaaaaaca gcaggctatc ttttcccact ttttgctttg tctatgtttg 49500 ttggacaatg tcaaactatg tagcccagac cccttctgta actttccatg tagaccaggc 49560 tggcctccaa ctcaaattcc cagcaatcta cctgtctctg cctctggagt tctgggaata 49620 aaggtgtgca tcaccatgcc tggcctcaat aagtccttgt cttatggtct tcttttctcc 49680 tccttactt cttatcctcc tccctctc ttccctttcc ctttatcttt atttctttat 49740 tgtcaccgtt tcttttctgc ttctcttacc atgctttaac aaccatcagg aagcaactaa 49800 ccaaatgttc tcatcttgag agtcaggtca gagatctag gagtcaagaa agacacgtgt 49860 aaaatagagc tcacatacca agcattaga aactcgcagt ggcactaaa gatggctcag 49920 gggataagaa cacttacatg gtgactcaca accattgta attccagttg cagacaattc 49980 atatctcct tctgatctct aagggcaccc agaatgtaca tacatacatt caggtaaaac 50040 gttcatatac atttaaaata ataagcctta agattagaa agagggctg gagagatggc 50100 tcagaggtta agagcaccga ctgctctcc agaggtcctg agttcaatc ccagcaacca 50160 catggtggct cacaaccatc tgtaatgaga tctgtgccc tcttctggtg tgcagatata 50220 catggaagct gatgttgta tacatattaaaaaaaa tcttaaaaa agagattag 50280 aaaaaaaa aaagctcat gtgtgtgtgg aaacctcat gaggtagaat gggataaag 50340 ctcttgattc tatgctt ctgctagat cacctctcct gttctctgga tctcttgac 50400 ttgactgtag aggttggctc tgtacaaggc agcactgtca gcagatcctg ttggttcaa 50460 gaggtaggtg aagccaagg actacaggtg acagcagat gggaggggccc agcacaggtg 50520 cagctcactg gaagggtcca gcacaggtgc agctcactgg aagggcccag caaagcgca 50580 gcaccttggg ctatagactt tgaccttgac caaatcactt tcctctctgc ttccttattt 50640 ctctccttac gaaattaaac aaataatcct catttacctg cctcacaagt tgtttagagg 50700 gatcaaatat tgctgttgct ttgtgaactg taagtagata agctatttag atgagatata 50760 tcatgattga cattcactca gccatgaagc acaggcttcc tgacaggcac tgctaggtga 50820 tggagagaa caaatgagtc tctccatctc tcttttctca gcacataggat tgaacccagg 50880 gccttgcata cactaagcaa gtgcactgcc gccgagctta tctgttttta cctcctttta 50940 aaaatattgt ttatgagctg ggtggtggtg gcacattct ttaatcccag cacttgagag 51000 gcagaggcag ggggagctct gtgacttcga ggccagcctg gtctgtagag tgagttccaa 51060 gacagccagg gctacaaaat aaaaccctgt ctcaaaaaac aaaaacaaaa acaaaaacaa 51120 aatcaaaaa atgtatgaat gcaaaaaacaa areatactt tagggtcgca ggtagattgg 51180 gaacttcaa tgtgggccaa aagaaggctg tggccctgct tctgaaggat ggtccagacc 51240 gatctcaga aggacaagt gtcggggcta agatgtagct tcgttgatag agttcatgca 51300 tgcctagcac gaatcctgg gttcagtctc cagccctgaa gaactggat atggtggttc 51360 acggcccgtaa cctcagcact tgggaggtag aggtagagg accaggatt tgggaccgg 51420 gcccagccta cgacgtga gaacctgtct tgatgatgat gataatggga ctggagagat 51480 ggctcctgtg taaagtgctg cataaacacg aggactgag ttctgtccc cagcaccagc 51540 atgataccag gccatggcag atataccctc tagctccagt atcgagtaga ggagacaggt 51600 ggaccctggg gcttgcctcc ccaccagctt agctgagaca gcaggctcta ggttcaatga 51660 gagatcctgt ctcaaaaaaaagagata atcaggaag acaccctggt gtcagctctg 51720 gcctccacac gagcctgcaa gcacacctgc ccatacaca cacacacacacacaca 51780 shit shit shit shit ccctggtgtc agctctggcc 51840 tccacacgag cctgcaagca cacctgccca tacacacac acacacac 51900 aaacacacac acacacacac acacacacac acacacctaa atttttttaa ttaatttaaa 51960 aagaagagac aatagcaata ccaagatgca atgacccagt ggacatgccc ctaactgtcc 52020 tcccatcgtg gagattgcca aggtgaggct gaagcagcta aagtctccat gaatgagagg 52080 aagctgtgag atgatgggga gggtccccac aaagcctgga gccttgtcag ccttttttcc 52140 tgcaccggaa accacaggaa accaggtcag tgtgtgtggc tgctccctct gctccagagc 52200 tcagctgtca gttttggggc ttcctgagca agctgtatta tttagtttgt ccttgaagct 52260 agagaccact ttgacctgga gtcgagaaga gttggaaagc cagctagctg gggagccaga 52320 gtgtacatgg ataacacaag aagtttattt gtgttatatg ttggcctatt tttttgccta 52380 actttaaatg atgtaccaat tgcttcttta tcttcagcat tcagtataga caagtgccga 52440 atagctgtta attctcaaaa tggaagatgt gtaactgtct atgacagatc caagttctag 52500 aacatgctaa taggcgtgcc agatctgcag tgtttgagga gggttaagaa atgggtgcat 52560 ttccacaaca gggtgctggt cctttcctg ttacctctcc gccacaccaa gagccccctg tgacgtgaga gcagtaaggc tttctaccaa acaggtggac aagtgaatta gtgttcacca gtgccctggc tcaggagag gaatgtgacc cagcctgctg gttaggatag aggaggagct 52740 tcagggcagg gtggatccag ggcagggctt ggtgagagcg cctgctggta ctgtgtgctc 52800. ctcacagctc cagggccagg ccctgctgcc ctgctgccct ctgcaaaca acaaagccat ggtctctgtg ccctgatcct gcagaagctt atggggagcc ccagactgac aacctggttc 52920 ctgcctctcc tcttactgct cttcagcctg tctatgtctg ctgaggttgg ctgtccctac 52980 ctgccacgct ggaccagcca ctgtctactg gcttcccatg tggtaaggtc ctgctgctag 53040 gtggggaca cttgcttggc acagagtaga aactccccag tctcctgtct gccctttcct 53160. acctctgaaa tccctgtgat ctagtggcag cctctctgtg tccaccatgt ggtcctgag gttctgttgg gctgaggtcc ttggaaggat gagaagaaaa cagctggggt ggatggacag tgggctgtgg gtctcagaac cggatgatcg gcacaaagag ttcagggagc atgcacctcc 53280. 53340. tgcagttccc ttctaaccac acctctctca tttcttttcc gcactgggct ctgtacctcc ttctgccggt gagtcctgtt tcttgttctg cttcactccc catgctcccc 53400 tgagttccag gtcccagtga gccatccatg gccattttgg ttcccagcct ggggagtagt 53460 ctagtatcaa tgggaaagtg cgggactagt gaagatttgc attgtaattc tgcctcagac gctatgactg cttttcactc tcccatctg ccctcggtct ctgagcacag gagaagagga gaatgatta tgcgatgagg acagtgttgc tctctaccaa tggggag aaaatgagca 53700. cctggctaa tctccaggca ctctggacaa gctggctctt cttatcaccg gtacctcact tgaccagga caataatcat ctttcatact ttattgtgca ttctgcttta ccaagctttc ccccatgcct tatgtcactt ggtccttatg acaaacccac tttgagcagt ctggcacctg cccaataact taaccaagat tgtacctgta agctatatca gagcccagcc cgaaatccag tttggagag gtaggtgggga ccccttaggc cctaccattt ttcttgttcc tctttttgtt 53940 tttgttttga gatagggcct cactatgtaa taacaaagcc tccataatta tttatttact 54000 tattttggtt cttcaagata gtgtttctct gtgtagtcct ggctgacctg gaactcactt 54060 tgtagactag gctggccatg aactcacaga gatccacctg cccctgcctc ctgagtgcta 54120 ggatcaaagg catacgccat caccacccag ctgcctccat cttttaatcc ttccatctca 54180 gcctcccaag gaatagctta cagccatgtg ttattgtgcc gactttctat catacacagg 54240 aagttctgtt caaactccct gcctaccct cctagccacg aagccttggc actggctctg 54300 ctgtttgtgc ctgcgtctga tgtcacggcc ttcaggtaag aaaagcagcc tttgagccat 54360 tcttagtgaa ggcatgaatc agggacagtt gaaaataact agctaggctg acacaagtgt 54420 caattgtcat attgggacat atatttgtct tgttaacctc aagagggact tagagcatcc 54480 tagaataaca catcatgaca gacattcatg ggaccactat atctttgtgg aggtgccatc 54540 tttcatctgg cactaattcc taaaagccat gtgttctgta gacttgctat tgttaccttc 54600 cctctgtaaa atgggactgc cactctgctg cttcattgcc tcacttggta catacggaac 54660 acacacttta aaaccagcag tcataggcgg atggaagat agcttagcca tcaaggtac 54720 tagatgctct tgcagaggac ctaagttcaa ttcccagcac ccatatcagg cagcatacaa 54780 ctgtaggtaa ctccaacct aaggaatctg atgtttctga ctcttaagc ctcattatta 54840 tgtgccaata catagatata catacacatg catagatta aacaataaat aaaatcacaa 54900 aaagttagtt taaaattgtt caccattagc tgagtgctag tggcgcacgc ctttaatccc 54960 agcactcggg aggcagcggc aggaggatct ctgtgattt gaggcccgcc tgttctatag 55020 aatgagttct aggacaggct ccaaacaat acagagaaac cctgtctca aataataat 55080 aaaaata aaaaaat aaaaaaaaaaaaaaaatca tgctacaatt 55140 aacactgctt tgtgggagg tgtgtgtgtt tgtgtgtgtg tctttttct 55200 ttgttttatt ttatttttta atatcttat gttagaggtg gagagatggc tcagttgttg 55260 agacagttgc tgccttcca gaggatctgt attagatcc cagtaccctc atagtggctg 55320 tatacacaat tgtctgtaac tgtagttcag ggaatccagt gccttcttt gacctcagca 55380 ggcaccaggc atgcgcatgg tgtacattca tatgtgcagg taaaaccttc atacacataa 55440 aaaaaaaaccta attattaaaaaaatgaa atatcttta tttattgttt 55500 gabagtctca tacatgcaga caatctatct tggtcaaac cacccacaat tcctccctcc 55560 catgcacttg gataactccc atacatcac tcatgtctt ttagatct 55620 tgtgtgtatg agtgctttgc ctgcatgtat atatgtgtat tacattcatg tagtactcat 55680 ggaagcatca aatccctgta gcactggagt tatagatggt tgggagccgc catgtggttg 55740 ctgggaacca atcctgaatc taagacatc aagtaggctg aactgctgat ccatctcac 55800 acacacac acacacac acacacac acacacac acacacgc 55860 gcacgcacgc accagagag ccatcaaatt ttgagacaga ggcttactgt gtagtcctgg 55920 ctcacctgga acttgcagag tagaccaggc aggtgtcaa atcaagagat cagcctgcct 55980 ctgcttctca atacaagtat aaaaccacgc ctgctctgt ttgtttgta aattcacgga 56040 gtccagctat gagtctgcct gctgggaatgt tgactgatct ggttggcttg accttgtgca 56100 gttcctgtgc aggtaaccac agctgcagtg ggttcatgaa cacgacagtc atgccatgtc 56160 tagaagactt caaaacactc ttccacatct tctggctctc acattctttc tgccctgtct 56220 tctgcatttg ctgaaccttg aaggggctta attctgtttt ttactgaatc ataatttttg 56280 ccaccaaaga agctcaatat cattgctacc cttgatttct ataaacattc tacccaacat 56340 tgggccacat ctccagatag gacaggggcc aagaatgccc aggtaagaag tgtgtttccc 56400 tcccacaggc cttcagtgga actggtttcc tctcttggtg aagaaatcta aaagtcctcc 56460 taagtctgaa ttctactgga ggcgtagaag gccagcatcg tcccaggtac cctatcattt 56520 acaacactat ggccctcagc ccctttcttt cttgcccctt attttgttct tcagcaacaa 56580 gagggagttt gaaaatgtgg agattacgca ctaagggaaa ctgaggccaa aggaatttga 56640 acaacttgcc tgttaaatta atttgtctct cagggttcag aggcagagtt agaaagcaca 56700 cttcctggat cctggcagac ttcctggatc ctggctcaca tccactttgt tctctgcttg 56760 cagaggaagc tgctaagcag cctttccctg tctcaggaag accatagcat ttccaacccc 56820 ttcccagcca tctcccatgg agggccacac tgcaaaagga cccagccttt ggacaccgga 56880 ggagtagaac atttcctag agcaggttca cagaagcgtg gaggtaggtg cgaggagcaa 56940 caagctggtg gtctcagcct tgcctgagcc agtctctgag tcgtccttct ccctaggacc 57000 tgaattctcc tttgatttgc tgcctgaggc acaggctatt cgggtgacta ttcctccagg 57060 cccagaggct agtgtgcgtc tttgttacca gtgggcactg gaatgtgaag acatgagcag 57120 tccttttgat acccaggtat ggagttccat gccttgcaaa gagacagatt acacaatttc 57180 tcagcaaatg ggatggggtg ggggggttgg ggcccagaca aggaaagtaa ttgtccaaaa 57240 cttccttaca aaatgata agtagcatta aaaacttgtt gggggcgggg gaggactaga 57300 tagaaagat ggtgggctat gttacttgag aagagagacc agaaagaaat gagaatggta 57360 tgtattgcca gggagaggac tcttggtgtg attcgggtca aattaagaga ctttgctaag 57420 ggctggggac atacccgttg gtagaatgtt tacctagcat gcttgatctc tggcactgca 57480 cagatggagt gggaggtgga agcaggagaa tcagaagttc aaagtcttcc ttagatatat 57540 aatgagtctg aggtctgttt ggagtacgtg agtcttaaga gaaaaaaaaa aaaaagctt 57600 tgtatttcc tcaggaggtt gttccctgaa aatttgacaa aactgtcata tgtgtcttgg 57660 tactttccc tttcatacct tttgtctagc cctcagaaat gatattcccc agtctcacaa 57720 tccaggtctt gacagcctac tttaactgcc actcctcaca gatgccattg tatgtagctg 57780 ggcatcacat gcagagactg gagtccttca gcttgcttca tgtctacaat ggctatacct 57840 tctaacaata ccagccttta gtagaattga tgcttctaat actgcatggc atacagctgg 57900 aactcaagaa atgtatcgcc tcttccctaa ccctactgct tgtagagtac ctcaagaaaa 57960 tggccaggat atagaaccta tgagagggag aaataagttc catggagtag gaaagtaagg 58020 tatattgtcc aaggtcccgg cttcatcctg ccttgtccat agtgtccatg cttttcctta 58080 ggtgtctctt tttctttccc tttgtggttt tatcaagaga aaattgtgtc cggggggccac 58140 actgtagacc tgccttatga attcctcctg ccctgcatgt gtatagaggt gagcacaggg 58200 aaatgtggat tagccatggc tccttgtcca accgcatctc cagtaagtca acttagaaag 58260 gactcgggaa ggatggagat gacttggtga tagagcaggc cgtacacact aaggagac 58320 tccaaaaaaa ccgggctact gggatgctta ggaagctggt gctggctagg ctatgagcca 58380 caccagaggt ggaagggtcc taggcaattc cacctcagcc ctgcctggat gctgtcttct 58440 ctggtccaga tttggcttca gctcacagac agagagtaga cagccatcat cgctgtgttc 58500 ttgagagcca aggttgcata tccctctcaa agtcatgttc tttccaaccc tccatttctc 58560 cttgtaagtc tgaagaataa gatgttttgg acatttttag ggggaagaaca ggtactaaaa 58620 gaagggtatg gacagagacc tgggaaacaa gaagctgaca gaggacaa ccagtggact 58680 ggactcgtag ggagtccaga gtgaggagtc aacacagggt actgggtact tgagaaagtc 58740 aaaatggag ctcagagagc ctaaaattga gtgatgcaac ctcgggaatt ctctgctgct 58800 gggaacagtt ctttggagac aggaggggtc atcccctctc atttgccact ccctcctatg 58860 ctctggaacc ccaacactta cacacactcc acagtgctgc ccattattgt ctgacagctc 58920 aagctgtttg atgacagtaa gccagagcca gaatgaagat gttaggaaag ccatttctaa 58980 caaacactct ctatgctccc caggcctcct acctgcaaga ggacaccgtg agatgcaaaa 59040 agtgtccctt ccagagctgg cctgaagcct gtgagtgttg tttgtattaa tcccgatgca 59100 cattcatact ccccctgctg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 59160 tgtgtgtgtg atggaggaag accccagatg gcaggaggac attttcttca ggagccaggg 59220 gaagatgcca cagctgtcag ccaacagatg agctcctgag tgctgaacat tatgaatgga 59280 aaaggatcgc ccagcgagtg tgtgagaggg ccaactgtag acctaggctc acttctcagt 59340 tctgccatag gttatcccca tagcccctcc cctttccctc atctgtaaaa tcccaaactg 59400 tttttgaggc ttgaatgaaa tattttaggg gaaagtggct ggtgttgtct ggagtgctgt 59460 atgtagaatg gtgaccttga ggagagagga cacttagaca caagtaacaa agtctaacgt 59520 acataggcag ggttatcatc aagggtatca gcccaggaca ggagaagccc agagacactg 59580 ttgtgagcag agagggtgtc acaggatggc acttgactga gctcaggaac tggacaggaa 59640 ttagaaagaa gaagcagggg gaagacagac tctgtgagga gcctacacag attctggata 59700 caggcgacag taagggctgt tcagtgtgga ggcctggatg gaaaggtaaa gccaaggaag 59760 ctgggtagaa agggagggag cgtggagtgc ttgcctgttg aggacctgtg aagcctggaa 59820 ttgtgctgga acattggtat tttctcctgg tgtcctgggg tggggtgggg agatggagtt 59880 acagctgggc tatggcctca ctcttgcacc tgccacctgc ccagatggct cggacttctg 59940 gaagtcagta cacttcactg actacagcca gaacaaccag atggtcatgg ctgtgacact 60000 <210> 3 <211> 9227 <212> DNA <213> Chinese hamster (Cricetulus griseus) <220> <221> misc_feature <222> (1)..(9227) <223> Expressed and VP-generating type-C group 1 ERV: ERV-C 109F <220> <221> misc_feature <222> (1334)..(1336) <223> Myr motif <220> <221> misc_feature <222> (1851)..(1868) <223> PPYP motif <400> 3 tgtgaaagac ccctctccct tagccctttc tttctcaagt ttgtctcctc ttcctcctgt 60 cggcggcttc cccgatcccc acccccggtg gcctttcccc gcccggcccg agaacaagca 120 ccgggtgggg ccggcccgag aacaagcacc gggtggggcc ggcccgagaa tgagcaccag 180 gtgggccagc ccgagaacga gcaccgggtg ggctggcacg agggcgagca ccaggtgggt 240 cagcacgagg acaaacaccg agtgagccgg cctggagctc tgcccctgag cccccgcccc 300 gcccgaagag aaacactccg tcccaaggtc tccgccccca aggtcagcca tcaggaaaag 360 ggggggaatt gagtctgctg taccagacac cagaccttga gaatatgctg atctggaatg 420 gctctgtgtc tcatttgaac catccaatgg aaatgattct gtatttcgcc tcatttgaaa 480 gactctgtgt ttcacctcat ttgaataact ctgtactttg cctcatttga ataaccctgt 540 atagcgcctc atatacattg accaatggga atagctctgt ataatgcctc attagaatta 600 tccaatagaa tccttgctcc tagcttgcgc cttttttcct atataaggac cccttttccc 660 ttggctcggg gcgcttagcc acacagaagc taagtcgccc caggtacctg cgtctccaat 720 aaagcctctt gttttacat ccagttcgtg gcctcgctga ttcctgggtg tgtgggtctc 780 cctctacgaa agtgcctctt cggggtcttt catttggggg ctcgtccggg attgagaccc 840 gcccagggac caccgaccca cgtctgggag gtaagtgttg tgcggatccg ctgttttgtc 900 ttgtctggtc tgagtctgtc ttgtgaattg cgcttgcgtt tgtagtatac agctgtgtac 960 atttgtaggc ggatccgagg agggactgac gggtccgaac tcccgaccgc ggctccagga 1020 gacgtcctgg tagcgtttga agccctcagg aagagggatt tgtattttga acttgggaag 1080 ccctcagggt gagagatttg tactttgaac ttagatctat gactggacat tttcccagtc 1140 tctttggaga aggccctcgg cttgagggat ttgcaatctt tactggggac gaggaaggag 1200 ggcccccttc ctcgactctc tctcaattc ttctgtcgac tctctgtcga aaccgcgctg 1260 cgaaagtctg ttctgtgtta ttcggtcttt gtcttgtagc tgtcatttgt gccctcctaa 1320 gcctagaaac tatggggcaa actgtcacca ctcctttgtc cctaacactc tcccactgga 1380 aagatgtaca ggaatatgct cataaccaat ctgttaatgt gcgtaaacgc aaatggatta 1440 ctctttgttc ttcagaatgg ccgacctttg atgtaggctg gccgcgagat ggtaccttta 1500 acccccagac tatattccag ataaaagaga agattatgga tcctggacca cacgggcatc 1560 ccgatcaagt ggcttatatc gtcacttggg aggctttggt tcaggacccc cctccctggg 1620 tacgtccttt cttacatccc aagggcccct cctccttcc cccctctaac cgctccaacc 1680 gacccattcc ttcggcccct acacctccca ctcctttgat tcctcccaac cccccttccc 1740 attccaacct ttaccctacc gtgatgaaag acactaaggc taagaaaag aagacaccta 1800 aggtactccc tccgggagaa gaccagttgg ttgatctatt aacggaggag cccccgccat 1860 atccgccact gccgcccccca ccagaggcag aagcggactc cgccgctgcc ttggcggaag cggcccctga cccttcacca atggcttatc gactaagagg tcgtagggag cagccccgttc 1980. cagattcaac cactctgccc ctccgaactg ggctgaacgg ccaacctcag tattggccat tctcagcatc ggacctctat aactggaaaa fatheratcc ttctttttct gcagaccccg tgaggctgac atctctcata gagtcggtac tcacgactca ccaacccacc tgggatgatt gtcagcagct tttgcaggtc cttttaacct cggagaga acagcgcgtg ctactagaag cacgaaaaaa tgtcccagga gtaaacgggc agcccaccca gctacccaat gaattgatg cggcttgccc tcttgaaaga cctgaatggg attttaccac cgaagcaggt aggacccacc tgcgtctcta tcgccagttg ctggtagcgg gactccgggg ggcaggacgc agacccacta 2400 atttggccca ggtgaagcag fathercagg gtgcggagga atcacctgcc gcttttctag agagattgaa agaagcgtac aggatgtata ctccctataa tccggaagat ccaggtcagg ccaccaacgt ttctatgtcc tttatttggc aatcagcccc ggacataaga aacaagcttc aaaggctaga aaatctacag ggatatacac tccaagattt gttgaaggaa cgagaacgta 2640 tttttaataa gaggggaaaca cagacagaaa gagagaacg ttggaggaag gaactcagg 2700 agagaga aagactaaga caggaagctg aggaaaaaga ggttgcgaga gaccgtaagc 2760 ggataaaga aatgagcagg ttattggcca cagatagtgac aggccaagaga cagaatagac 2820 agagggatga cagaaggggg ccccacctgg acagggacca atgtgcttac tgtaaagaaa 2880 aaagcattg ggcaagagaa tgccctaaga accccccgggc caagcttcca ccgccaaggg 2940 tttctgacct cctgaaccta gaagattaga ggagtcgggg ccaggagccc ccccctgagc 3000 ccaggataac actgcaagtc ggggggcatc cggtcacctt cctagtcgat acaggggcac 3060 aacattccgt tctgaatcgg tcaccccggac ccctgagtca caggactgca tgggtacagg 3120 gagctacagg cggaaagcag taccattgga ctacaaatcg gcagctccag ctcgcgaccg 3180 ataaggttat gcattctttc ctccatgtgc cagactgccc ctacccctta ctaggacggg 3240 acctattgac caaattaaaa gctcaaatac actttgagag gtcagaagtc aaagtcacag 3300 ggccagagggg aattcccctt accatcttga caatgtccat agagatgaa tatagactcc 3360 atgaaaagg gactattcg aacaatcagg aaacccttga tcactggctt gcggaatttc 3420 cccaagcctg ggctgaaca ggaggaatgg gccttgccat taaccaggcc ccaattatag 3480 taaccttaaa agctgccatc cttcctgcat ccgtcagaca gtatccaatg cctaagaag 3540 cccgcgaagg aattcggcca catattaaaa ggttacttga acagggatt ctggtgccct 3600 gtaaatctcc ttggaataca cccttgctac ccgttaggaa gccgggaact aatgactacc 3660 ggccagtaca ggacctgaga gaagtcaata aaggataga ggacatacac cctactgtcc 3720 ccaaccctta caatttgctg agtggattgc cacctaacta tacctgtac acagtcttag 3780 atcttaaga cgctttcttc tgcctccgcc tgcatcccac cagccagcct atatttgcct 3840 ttgaatggca ggacgcggcc cttggaatct ctgggcagct gacttggact aggctacctc 3900 aagggtttaa gaacagccct accctttg atgaagcttt acatcaggac ctggcagaat 3960 tccgggttag gtaccccgct ctaatcctct tacaatgt agatgacatt ctcctggcag 4020 ccaaaaccaa agggaaatgc aaggaaggca ctcaagccct cctccagact cttgggagcc 4080 tagggtaccg ggcatccgcc aagaaggccc agatatgtca gaaacaggtg acctatttag 4140 gatacaagat aaaggatgga cgtcgatggc taacggaagc ccgtatgcga gccatcttag 4200 acattcccac cccacaaaat ccccgccaac tgagagaatt cttgggaacg gcaggcttct 4260 gccgcctatg gatccctggg tttgccgaaa tggcggctcc cctctacccc ctcactcggc 4320 caggggttgc ttttaaatgg gaagagcccc aaaagaaagc cttcaccgac atcaaaaagg 4380 ctctccttga atcaccagcc ctgggtctac cggacttagc taagccattt gaacttttta 4440 tagatgagaa ggagggctat gctaagggag tcctcaccca aaatctgggg ccttggagaa 4500 ggcccactgc atacctctcc aagaaattg atcctgtggc atcgggatgg ccaccctgcc 4560 ttcgaatgat tgctgctata gcctgctgg taaaagattc tcacaagcta accttggggc 4620 agcctttgac catacatgcc cctcatgcag tagaggcagt catcagacag cctccagata 4680 gatggcttac taatgcccga atgactcatt accagactat gctgttagac aaagaccggg 4740 tccacttcgg gcctttggtg actctgaacc cagccaccct gctccccctc cctggggagc 4800 ccgaggctca tgattgctta caggtattgg ccgaggccca tggagcgaga tccgacctga 4860 ctgaccagcc tctacctagc ccggaccaca tctggttcac ggatggaagc agctttttgc 4920 4980 aggcactccc ccctggaact tccgcacaga gggcagaact catagcactc acgcaggctc 5040 taaaattggc agaaggtaag aggctcaccg tgtatacaga cagtcgttat gcctttgcca 5100 ctgcccatat acatggagaa atttacagac ggagggggct gcttacctcc gaaggggaag 5160 acattaaaaa tagggaggaa atcctcgctc tcttaagggc tcttcatctg cccgctgcct 5220 tagtatcat acattgccct ggacaccaaa aaggggattc tttcgaagca aggggcaatc 5280 gaagggcaga cttggctgcc cgagaggcgg ccctgaccac agacaccact aacctcctgg 5340 ctctagagcc caccaacgac catcccttcc cctcatggga ctatgaacaa agagacatcc 5400 aaaccctaga gaattggga gccgcaaagg aaccaaacgg ggattggact tatgaaggaa 5460 agactgtcat cccctaccgg gtaaccaagt acctagtgac atttcat aagatgacac 5520 atctgagctc caagagatg cgggagctcc tcgaacgaga agaggaattc atttccttt 5580 tggggaagaa cgatattcta aaacaggtaa ctgagcaatg tgatgcgtgc gcccgagtca 5640 acgcatccag actgaagctt cctcccggga accggtcag aggctaccgg cccggacac 5700 attgggagat agatttcact gagatttaac caggaaata tggatacag tatctatta 5760 ttttgtaga cacctttca ggatgggttg aagccttccc tactaaacat gaacagcca 5820 agatcgttac taagaaattg cttgaagaaa tctttccccg ttatggtg cctcaggtat 5880 tgggaacaga caatgggccc gccttcgtct cccaggtaag tcagtcagtg gccaccttat 5940 tggggattga ttggaaatta cattgtgctt atagacccca aagttcagga caggtagaaa 6000 ggatgaatag aacaatcaag gagacttta aaattgtc gcttgcaact ggcactagag 6060 actgggtcct cctactcccc ctagcactct accgcgctcg taataccct ggaccacatg 6120 ggctcacacc ctttgagatc ctgtatggag tacctactcc tatcattac tttctgatc 6180 aagatgtctc agattttgct aactcccctt ctctccaagc tcatttacag gccctccaac 6240 tagtacaacg ggaggtctgg aaaccccttg ctcaagctta taaagaccag agggaccatc 6300 ccaccatccc ccattcctac cagatcgggg acactgtttg ggtccggcgt caccaggcca 6360 agaaccttga accccgctgg aagggaccct acatcgtttt gcttaccact cccaccgcac 6420 tcaaggtaga cggcattgca gcttggatac atgcttcaca tgtaaagcca gcccaaccca 6480 ccgattcagc cactgcatca gaatggaccg cacaccgcac tcaaaatcct ttaaagataa 6540 gactctctcg tacaccctcc tgttgattg ttgtctgttt accccccatg tagcaactaa 6600 cccccacagg gtttataata tcacctggaa aatagccaat ctagggaccg gggaaatagc 6660 caacctcagc acttatatag ggactctaca tgatgggttc cctcctctct atgtcgacct 6720 atgtgactta gtagggtctg attgggatcc ctctgaccag gaaccattcc cagggtacgg 6780 atgccaccac cctgggggaa ggataggaac aagaagcaag gatttttatg tttgccccgg 6840 ccataaacca actcatggct gcggggggcc gcaggaaggg tactgtgcaa gatggggatg 6900 tgaaccaca ggggaggctt actggaacc ctcttcctct tgggattca tcactctcaa acggagggag atcccagggt acgcaggga aggaccatgg agatgtgggc aaagagcctg 7020 cggaccttgt tatgatagtg ccggaggggg aggttttcaa ggcgccaccc ccggaggaa 7080. atgcaaccct ctcatcctaa ggttcacaga tgctggaaaa agaactactt gggatagtcc taaggtctgg ggactcaggc tgcaccgagc agggaagat ccggtgactt tattctccct gtacagacaa attactcccc tagcacaca atcagttggg ccaaacatag taatagcgga ccagagatcc ccaacccatt ttcaagtccc taaccccct accgttccta aagctatcac 7380. tcctacacca ggtgctgtca ccttctcccc caccccagat gccctaaaca tcgagataac cagagaccct ccaggtacca gagatagatt attack atccaaggag tttaccaagc cttaaatttt tcagacccca acaagactca ggaatgctgg ttatgcctag tttcccggcc cccatattat gaaggcgtgg caatactggg caactactcc aaccagacct cagcacctac cagttgcgga gctgctatgc agcacaagct cacaatatct gaggtctcag gaaaggggct atgcataggc aggattcctt cctcacatca agaattatgt aaccaagtag agccattatc 7680 tcaggacagc cgataccttg ttgcccctta tggaacttat tgggcttgca gtactgggtt 7740 gactccctgt gtctctacca ctgttctcaa caccaccatt gactttgta tattgataga 7800 actttggccc aaagtcacat accaccaacc tgaatatgtt tacagcgtac tagagaaatc 7860 aacccgatat aagagggagc caatatcctt taccgtggcc ctattattag gaggaataac 7920 aatggggggc atagcagccg gcatagggac cggaaccgtt gccctacagg gaattaatca 7980 ttttaagctt ctacaacaag ccatgcacac ggatatccag gtcctagaag agtcagtcag 8040 tgcactcgag aaatccttaa catcactctc tgaggtggtc ctgcaaaaca gacggggatt 8100 agatttatta ttttacagg aaggggggct atgtgctgcc ctcaaggaag aatgctgctt 8160 ttatgcagat catacaggaa tagttaggga tagcatggcc aaacttaggg agaggctaaa 8220 acagaggcaa cagctatttg agtctcaaca aggatggttc gagggatggt tcgctaartc 8280 cccctggttg actaccctta tatccacgct catgggacct ctggttattc tatttttgat 8340 cctcatattt ggtccctgca ttctgaacaa actgactcaa ttcatcagag aacgactatc 8400 8460 gtatctagag acctctgaat gaagattcc attcagttac aagagaaatg ggggaatgaa 8520 agacccctct cccttagccc tttctttctc aagtttgtct cctcttctc ctgtcggcgg 8580 cttccccgat ccccaccccc ggtggccttt ccccgcccgg cccgagaaca agcaccgggt 8640 ggggccggcc cgaagacaag caccgggtgg ggccggcccg agaatgagca ccaggtgggc 8700 cagccccgaga acgagcaccg ggtgggctgg cacgagggcg agcaccaggt gggtcagcac 8760 gaggaaac accgagtgag ccggcctgga gctctgcccc tgagccccccg ccccgcccga 8820 agaaacac tccgtcccaa ggtctccgcc cccaaggtca gccatcagga aaaggggggg 8880 aattgagtct gctgtaccag acaccagacc ttgagaatat gctgatctgg aatggctctg 8940 tgtctcattt gaaccatcca atggaaatga ttctgtattt cgcctcattt gaaagactct 9000 gtgtttcacc tcatttgaat aactctgtac tttgcctcat ttgaataacc ctgtatagcg 9060 cctcatatac attgaccaat gggaatagct ctgtataatg cctcattaga attatccaat 9120 agaatccttg ctcctagctt gcgccttttt tcctatataa ggaccccttt tcccttggct 9180 cggggcgctt agccacacag aagctaagtc gccccaggta cctgcgt 9227 <210> 4 <211> 30019 <212> DNA <213> Chinese hamster (Cricetulus griseus) <220> <221> misc_feature <222> (1)..(30020) <223> 5' of SEQ ID NO: 2 <400> 4 tggttctatc gagacagcta catccgcttc tccacacagc ccttctccct gaagaacctg 60 gacaagtgag ctttccctcc acctcccttg actgcccagg ctagtgagga ggtcagcaga 120 caaacagaac agtgggctct gcgggcaggg aaggcaggct tttcgccgcc actcatcctc 180 tgccctgaag gagcttgcca agagggtgcc cttgggttac cgaaatgagc aaaacaagaa 240 ctcctgctaa tcaagggctt acatcctagc aatgagcttg agtaatcatg gctaacatgc 300 tagaaggtga tatgtgttag gaagaaaatg tagatctggg taatggcaga ttaagagaaa 360 cttgagaggg gaaaggcaac ttgcagtttg ctcttgggat gagaggaaac cttgaggaga 420 cctgggttca aattctatct ctgcagcatc cagattgtgt tcctctagag acatgaaaag 480 agacttaagc ctcagtttca gttagagccc agtggtccca tctgtaatcc cagcacttgg 540 gagtctgagt ctgaaggatt ttgaggtcat tctgggctat ataatgagac ccccactcaa 600 gacaaaacaa gccaggtgtg gtagctccta actgtaatcc tagaacttgg ctaaggcagg 660 agccaaaatc aagccaggta cggtattgga ggcccgtgat cccagtgctt agaaggtaga 720 ggcaggacaa tcagttcact gtcaccctca gctacatgcc aagttttatc taacctgggc 780 aacatgagac catttcatag aaaaagacca gcctccgttt ccacatctga agactggagc 840 agcaatacac tgtagctgct tatcatcaag tacttcacat gtgataagtt gttcctgctt 900 ggttatacca cagccacttc tcaatggctc acaatgaagt gagttattgc cattttaaaa 960 agagatgaga ggggcaaagg ttaaatgact tgcctgagat ctcatagttg atcatcggtg 1020 aagctgggat ttgaccatcc agggtcatcg tccccctgcc acacatagga atggtgaact 1080 gaaactaggc ccaaagcact tgatacttac tgtggcctgc aggcaacact ggagcattct 1140 gagaaatgac tagccctggc cgtgggtgaa tagggagtgg ggcagggagt gggggtgggg 1200 tggaggggtg cagcttctgt gcttctgtcc tgttgatctt ccagcagggg cccctctcag 1260 ttgagatccc ctagacggct gaccacaggg ttctgcctgc agccctgagg ttgggctcca 1320 gggctgagcc tgtgtctctg atccactcta gctctgtgca cctatgtaac aattccatcc 1380 agagatacct ggagacctcc tgtcaccggc accggatgct gccctcggac aacatgtggt 1440 ccagccagag gtttcaggcc cacctgcagg aaatgggtgc cccaaatgcc tggtctagtg 1500 tcattgtacc cggcatgaag gctgctgtga tccatgccct gcagacctcc caagacactg 1560 tgcagtgccg aaaggccagc tttgagctct atggggccga ctttgtgttt ggggaagact 1620 tccggccctg gttgattgag atcaatgcca gccccaccat ggcaccttcc acagctgtaa 1680 ctgcccgcct gtgtgctggt gtgcaagcag ataccctccg tgtggtcatt gaccggcgac 1740 tggaccgtac ctgtgacacg ggagcctttg agctcatcta taagcaggtg agatgtccca 1800 gcacctccca caggcaaccc tacagcaaag ccctggctgg ggtctgctgt gagacagagt 1860 tcaagactga ctctacacac ggggcatctt aacacagaca cgtcccactg gcctgtctcc 1920 tcatctgtgg aagatactgt cctttgagag ccattaatgc catgagtttg tagtcatagg 1980 tagtattttc agaggccctg ggacctgctc agtttccatg gtaattccaa gcctctaggt 2040 agtaaccta ttctctcatc tgtaaagtaa gactgtacct ggctcctcct ttgtgtgctg 2100 tgagaatggg tggttgatc ttcacacaag ggtttgctaa agtaccaagc tggaggacat 2160 agaggatatg aggccatgaa cctcaaggcc tgcctatcaa gagttaatta ttagtgtcta 2220 tcattgtat aacgattatt atgttactca tcttcttcca aaaacagatt tgatcttgct 2280 tatttataat aagtatagaa ttgcttgggg tttttgtttt tgtttttgtg ggttttgttg 2340 agatagggtt tttctgtata gctttggatc ctgttctgga acttgctctg tagaccaggc 2400 tggccttgaa ctcaccttg actgcctctg cctcccgagt gctgggacta aaggtgtgca 2460 ccaccattgc ccgacctaga attgtttttt attcagccaa aaaacattac cttgccctcg 2520 tggtctaatt ctctctagaa acacccttag gagcacacca ggggcatgcc ctatagaaat 2580 cctaggtttt ctcagtccag tctagttgac aactgatatt agccaccaca gctggacatg 2640 gggctccacc ttccacctcc cagtatttgg aaggctgaga caggggatca cttttagttc 2700 aagggaagct tgggttacac agtgagttcc aggctagcct gggcttcaca gtgagaccct 2760 acttaaaaac atcatacaaa caaacaaaca aacaaacctt taaacgtatg tttttaaggg 2820 tttctgaatt ctgaggacag tttttaagat ctttgagcta agattcacca aggtctaggt 2880 ttgctacagg aagggaaaac actgatcacc tgacctgggt ggcctcatgt ttcctacagg 2940 tcctgatcac cttagaataa tgtgctggca aagggttccg tctttgttag ggatgccatc 3000 actaggtgtc cagggtggaa tccaggcctg cgtttttagc atgtgcagtt ctactgagct 3060 acacctccag cctaaaaaat gtaaaggagg agatgcatgc aggtgtgcat acacctgtaa 3120 tccctgggca acagaagcaa gaggactgtc acaggttcac caccacctgg ttacagggtt 3180 tataataagg tcctgtcaca aacaatgtag tcttggaaga gaggagagga aatggggaag 3240 aggaagtaat ttgcagttta aaatagagca aaattgccgg gcgttggtgg tgcacacctt 3300 taatcccagc actcgggagg cagaggcagg cgcatctctg tgaattcgag actagcatgg 3360 tctacaagag ctagtttcag gacagcctcc agagctacag agacaccctg tctcaaaaaa 3420 aaaaaaaaaa aaaaaaaaaa agcgaaattt gtatggacat ggcaacacgt gcctgtaatc 3480 ccaacactta gggggctgag gttggaggat caggagttca aagttatcct tggctgtatg 3540 tgagcttgaa gccaacctgg gcctacagca ctgtctgttc cacaatctgt ctttatctgt 3600 ttgtctcctt atgttgttca gctcggtctg ttctctgaat gtttgtctca gaacaaacaa 3660 aattgaatag ggctgggcat acgcactttt caaatgtcct ttgtccccca ggtatctttc 3720 attgctgatt tggttttgag cttgagggtc agtgtacaac aaggtggtta caatggtggc 3780 tttgtctgtc tctgatctcc tttatctagg acagtgccac tgctgtatcc ctggcaccct 3840 ggtcctttgc aggccaggca ggccagcctg caccaccgcc ccacactgtt ttatctgttt 3900 ttctccttat gttgttcaga tcggtctgtt ctctgaatgc cctgtaaact agaagatagg 3960 ctaacaagct gatggggttc agggttgaga tttttggcaa aaaacactca tgtgatgcta 4020 ggtacctcat gtgactgtca caaggcacaa ccaggaatct ctagttccca atgccgaatt 4080 tgaccttaga ctaaggtggc tgccaccaga gccacatcct cccctttgta gcttttttca 4140 tttttcttta cattatttat tgtgtttgag tttgtacatg tgtgtggtca cacatgccac 4200 agcacacatg tgggagtcag agggcaactt atgggagttg gttctctcct cccatcccat 4260 gggtcctggg gcttgaactc agcaagtacc ttataagcta tctcaatact gtttgcctcc 4320 aaaatgtatt actttgtgta gctgtccctt ttctgttgct aataacagaa tactacagac 4380 agtgtaagtt acaaagaaaa taggctcaat tgtatccatc ttttgctgcc catggcatga 4440 aaacacttgc atcccaacac aggccagagg tcgctttagt gtgaagcagg attgagtgca 4500 tgtctgcatg gctaagactc tctacttctc tggtttcctg atccctaggg ctctgggact 4560 ctagatcctc tggacccctt gataatagag ggagcttcct gtttctcaga agtgcctttt 4620 gaccatatat cccagaaact gattccatcc atctctgccg tgtgtcacta gtcactagat 4680 ggcgtcactg tcatctctca ctagtgtctg agatggcctt gtcttctctc ctgcccacag 4740 cctgctgtgg aggtgcccca gtacgtgggg atccggctca tggtggaggg ctctaccatc 4800 aagaagccca tagcagcttg tcatcggcgg acagcggtcc gctcatcact ccctcatctg 4860 ctggcccagc aaggctgtgg ggaaggcaag gactcaggac cccctatcca caggtcagct 4920 tctaggaaag atgctggggc caggagcctg ggacacactg agaagccaga ctctgcggcc 4980 accacctcag tccccggaaa ggggaagaaa ggcaaggcaa aaagtgccac agccctggtc 5040 tgcatcaccc tgcagaaatg ggagtcccac aacaccaggg tgggccccac cttcaacagg 5100 ttaatgtgtc tgaaacagcc tgaggcctgg ggtagtacca tgtcccccaa accccgcagt 5160 gttcccaagg ccatttctgc ctgctctcca agccctcccc aagcatctgg gcttgccctc 5220 ctgccaaaag gccaccagtg atagcaagta tgaaccaaat atctttaaat acataaccaa 5280 atgagtatta caaagtagtc accctgccag gcagttagac caaaggctcg gtcctagagt 5340 gcgcgcccag agtccagacc catgctgctg ctctagccag cctttgccct cacctttctc 5400 tggagaaagg tgctgccacc atgcccttcc ccattcctaa ccagccccct cagccctcat 5460 aacgccctag tgaggtaggt gctattgtcc ccattttcca gccgaggtag cagcaagttt 5520 aaggacgttg cccgaggttg cacagctcag aaggggcaga gctgggatgc agacccaggt 5580 ctgttggtct cccaaccctg tgttcttccc actgcctctg gaggaggagc tgggaggggc 5640 tccatctgcc cttaccttgt atcccccacc tttacacatg tactgtggaa caattggtca 5700 ggctggggcc tcacccagat cctcacagct tcccttctcc cacagccccg tcctacctcc 5760 ctagtctcca ttccaaggcc tggctgcctt cttcccatgt gctccgaccc cagggccggg 5820 tcctcagact accgaatggc caactggtgg gctctaaggc tctgtcaacc acaggcaagg 5880 ccttgatgac tctacctact gccaaggttc tgatgtcctt cccacctcac cctgatctca 5940 agctggcacc cagcatgctg aagccaggaa aggtgggcct cgagctgtgc ctcacaccct 6000 ggcgggtagt gctgagcagt gggatcgggg ctgaagggca cgaacagagg gcagcgctcg 6060 gaccatacag cgccccaggg aagggcttgt cttctccaga accctgttcc aagacagagg 6120 cctgatcata tctcttccc tccctcctt gcaccgaggc tgctattccc ctgcaccttc 6180 gaggccccca ctttggaagt gcctcgaggc ctctgcctt tgaagttgga cctctccta 6240 gcaccacag gaaagtcacg gccaaggca agttcaaggc catactctgc gawaagcca 6300 gggctgaggc attackccaag aagaggctga gcctccccaa acccttgacc cttattctga 6360 catgccggac actgagaacc atggggta ggaggctaga gaaacccctg ctctgatctc 6420 tactgcccca tcctggatcc agcatcaat taaaaaagc aattaaagtt ctctggactt 6480 ggcttgaata atgtgcggct aggctcataa aagagttgac cagcagggcc tccatcagca 6540 agggccacag tcccaccca gcgacagaca ttggctttct ctgcagggag acggatggtt 6600 ggggaaagag ccttcactat acgatgatg acactgagac atggcttgcc tgagaccaca 6660 gcaggcggaa agtcagccat caggagtgcc ccttcccaa vakaagctgg gctggcagaa 6720 gagcttctga tggtcacaga acatgac agagacggg ctttccctaa acctcagcct 6780 ttctccggag agtatgtccc tcagtgaggg gtcggtcaag acacctcaa ctgcaaacc 6840 caagaaaac gtcaagtgtg gcatttccta cctgtagagc ctcagctgct ggtcctccaa 6900 aagcggtatc taggcttagc gtgtgaatgc ttcctgagtg gggcagggtg gaggaggag 6960 tgcggtgttg aaatccaatg acctgtgtct cccaaagtca gaagagctca tgcctgcaca 7020 gtggtgtgtg tctgtcctcc caggacttgg gaggcagagg caggtgcatc tctgaattcc 7080 agtccagcca gggatatacc aagaggccca gcctcaaaag caaacaaacc tcctcgtgac 7140 caggagatca gcagatgcca cctccagacc tggccactgt tcacttgagc agagagcaca 7200 gtccctggtc aacacttgct ttctcagcag atcctcaaaa gcagacttgt gagtaggac 7260 ttttattatt ttaagtccag agagcggtca cctgcccaac gccacacagt aagtgagtgg 7320 tttaactggg atttatctta ggttggtagg actaagggt caaagacctt gaccgtactc 7380 agcaccaagc ccactgtcct tgagctgggt cacggccctt ctttctattt tccaattggg 7440 ggttaagcac tgtctatcgg tgagagagcc gcaggcactg cagggtccga gagagatcg 7500 aggtagggg tgccacaagt tcactagggg ggtccctgga tctggtgcct gggaggagga 7560 ggcggtgcaa gtgcaggtgc aagggcatct gggccacctg ggaaggacgc aggcgaaggc 7620 gtctgaggag agcttcgtcc agcacctgga gtgggaaaga cagcagccca cctgagttcc 7680 agccagagga gcccctggct ctgatggacc tctctggtct gcaactgcca tcattttctc 7740 aacaggcagg cagggatttc tctccacaca gagctaagtt acgtttcagc tccttttgtg 7800 tttagtgaag ccatgtgatt aagccactca ccaatgggat gtgaggaaaa cacagaccta 7860 gccctcaaag ccccgattca caaaaacatc cagtctcccc ttatggccag gaagcaatag 7920 cccttttctc caagtggcta cccagagtca ggtccactcc tactgtatgc ccatttgcca 7980 gacttaatcc aagaagaaac aatggacttc agggcctgtc agaggtcagc tccccactcc 8040 ttgagctaac agacagaagg aagcctgagg aagtcacagc accacagagg caagggtggc 8100 cccagaggcc aagcctgttc ccccaattcc ctaaatacag agcaggcatg tgggcctgaa 8160 gacctacctg tctctggagc ttggtggtct tggctggcca cagaaagcgc tggcccagaa 8220 cggtgcccac tcgaggagca taggtgtggt ccccaagcac tgggcagagc tgtagagcca 8280 tgtgcacctg aagctgactg gggaacactg aaagatgggg ggaggcagtt gctcttcttg 8340 atcatcaagg actgtcccca ttcccagagc ctgtgcatct atgaggtgac ctctgcctcc 8400 acagagggcc atgagcagat caggacaatg acagctggca accttcccag cctgggggca 8460 ttgaacccaa cagaactgaa gctcctggga ataagtatgt gggttgggtg ctaaactgtg 8520 ggtgtgcacc taaaactctg cctcctgctg agcccactct cagagtcccc agatgctctt 8580 ttttttttgt tttgtttttt gttttttcag gcggggtttc tctgtggctt tggaggctgt 8640 cctggaacta gctcttgtag accaggctgg tctcgaactc agagagaggt gcctgcctct 8700 gcctccccag tgctgggatt aaaggcatgc cccaccaccg cccggccacc agatgctctt 8760 tggatgttgc aaaacaacat cttcctcata ggcttcattt cccctgcaga gtactgttca 8820 gccctacctg tcagtggctg cagctggacc agagcacagc cacagcctgt ggccatcaca 8880 tggaaatggc tgagggtcct cttgacacct tctaggatgt cctttcgaga tggggatgtc 8940 acggggatgg cctaggatac cagtcaagaa tgacttagca cacacgtagg agcccaattg 9000 agccacccct gctgccggct gacttaggac agggcctcag aacaggcagc agcaacttgc 9060 ctgttagagg acaggggaat agccacccca tgcacatagt gcagactcct ctgtaaggca 9120 aaacctgaaa ggactcctag tggcttctga ctcacaagat caatgccatc catgcgttcc 9180 agcttcaggg ccacgtggat agtcccctca gaaggctcag ggatgccatc agtgatgcca 9240 ctgagaaaga gaaaggaagt ctcagagcag ccagctggga ccttccttgg ccctgggagt 9300 caccccgcat ctctcaccag taggtggctg tgggcctctg tgttctcctt gagtgaacga 9360 agaacttctg caagcggctt gctgtctggg ggcagctgga gagaagcaca agcccagacg 9420 cctccctgaa agaagaggac aagtcacata aagcctcttg gttggaaaaa atgagggcca 9480 gattggctgc tcctgcagag gactagcact cggcacccac atagtgggtc ccaactgtaa 9540 ttccacttcc aagagagctg atgccctctt ctggcctctg gggcaccggg catgcactgt 9600 ggtacacaga cacacatgca ggctggcctc cgggggcaca aggcatgtgt ggtgcacgga 9660 catacatgca ggctaaacac acatttaaaa acaaatcttt ggtcttttttt aaaggagacc 9720 ctcccaccaa ggggctggag aaatgaatca gtggttaaga gcactagctg ctcttccaga 9780 gtacctgggt tcagcacca catggcagct cacactgta gctccagttc cagggatct 9840 gagaccctca cacagacaca catgcaggta aaacaccaat gcactatata tatatatata 9900 taggataga tagataga tagataga gagagaga gagaccac cttcccacct 9960 ccccaaatga acaccaggac tacagtccag acctaagaaa caaatcctgg ccaggcagtg 10020 gtggtgcacg cctttaatcc cagcacttgg gaggcagagg caggcgcatc tctgtgagtt 10080 cgagaccaac ttggtctaca agagctagtt ccagggcagc ctccaaagct acagagaaac 10140 cctgtctcaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaagggggg 10200 ggaataaac aaatcccaag accttttttc tggcccccag agactcaga CAATGCCct 10260 aagagttact accatgcca ggcacctgaa taggactt atacagtg ggtgtaaaat 10320 aaagcccac cacactccctc acttacttc caggtgctcg cacaacctgg agctcccggt 10380 gtttgagcccc cagggcctgg ctcagctgtg gcagcacaga aagcaggtc agctccccag 10440 gtctccctga gaagtaagag ggagaaaaca agttgccaga gcaccatcca acgaactcct 10500 ccccaccagc ctttccctcc ttgtccctgc ccatacctgt cacaggcaga ccctgtggct 10560 tgttcagtgt caccagaggt cctgaaatat tcggtagatg tcagcaaaag cagggcagcc 10620 tgtgtgcatg cagcctctgg ttataagctc ccaagcccac tccaaactga acttcctgct 10680 ccccgcaccc agggcctttg cacctgctgc cctgagcttt cattaatatt gggtccctta 10740 tcacacacct ccctataatg tgtgcccctc ctacagtcct tgactgtttg gtctgagacc 10800 agctcaaaca ggccggcctc aaactcaact ataggtaagt ctggtcttga actcttgatc 10860 ctccttgcct ctgtctccca agttctggga ttacaggtgt ggtcaccact cccagaaaca 10920 gcagagattc actattcctc acatcagaac gcttgctctc tcaaggcagg agccagccat 10980 atttatccca gtactgtgta gcctcctcat tgctacaatg cactgaagtt cagcaaagtt 11040 gtcattccta aaggcacaca gcatgctgct tacagacccc aggaaactaa cttctgctcc 11100 cgagggtctt agaatgcgag gcggtgtgaa ggtttcctat gcctggcgga gggaatgtga 11160 cggctacagg ggtccgactc cagggggtcg gaggctccct caccttgctg atccaccaca 11220 gctgccctca gcacctcagc cagctcctcc gggccgaggt tctctgttcg cagaagcccg 11280 gggaagggct ggtcctccac tgcgtccttg cacttgctgg aaccttgagg ttgaggccga 11340 cccctgagaa aaacgacaga agccggtacc tccaacgccc acgctgccca gtgcagcctt 11400 ttcgtacaca accctcagaa cccccggctc ggctgacagt tgtggtccac aggcccccaa 11460 gactacagaa ggggcatcag acgctcggag cgccaaccat ctcgtgtcgg gtcacacaac 11520 gcggctggga ttagaaccca tgtcctgatc tgagtgcccg acctgccctc cccttgtcct 11580 agcctccctc ctacacggaa gtgttgagtt ataatcaccg ggcctcggtg ccgaagcctg 11640 catctctcgg ccgtgcgcca gccgccagcc ggggccgcca ggtgccagac acacaacgcc 11700 aaacgcggaa gacgcccatc gccgcagcaa gcacagacgc atgcgtgtcc acggactgcc 11760 ccaccgcgtg catcctccgt gcgccgattg gtcagcctgg ctgtcaatca gagcatcgag 11820 caggcggagc ttcgaggacg gaagagccaa actttcctttt ggctgaggaa ggtaaagtac 11880 ggtctccggc tctgctttgg gggaaactga ggcaggaggc gcggttattt actcgcggta 11940 gaggacgtgt cttagaatag aaagaggcga gtttgcagat agattgtttc agttctcagg 12000 acccttaatc tgaaacccat ccttgtaaag cagaggggaga aggtgaagcc ccacagcctt 12060 cgggccaggg cacctgcatg gaaatggtct gcgacattaa ccatcccatg actgtggctc 12120 aggtggtaga gagattttc taggatgcac gagttcctgg gttcgatccc cagcaccaca 12180 cgaagaccaa gatcacagat acagactgag ttggagacca gcctgggata tgtaaggccc 12240 ttctatcaca agaaaaacaa actaggccgg gtgtggtggt gctcttcttc aacagaggcc 12300 aacctggtct agacagtgag tttcaggaca gccggagcta cacagagaaa ccctgtcccc 12360 cccccccaaa aaaaaaagaa aagaaaaaca agcttggtgc ggtccacata gtaagttcca 12420 cactggccag tatgttgtta attccaaaga tggaatgagt gtaaggaaaa aggccagcca 12480 aagaaacaca ctttggctct gcaaccagaa ccaaagaaat gcacccttgt cagaagccag 12540 ccttggtgac gcccccccccc ccaatgccta gctagccgcc agctagccta gcatcccacc 12600 cctcaggttc caggacctgg cctgagaagc aactaac...

Claims

1. An engineered CHO cell, characterized in that, The engineered CHO cells contain within their genome at least one transgene encoding at least one transgene expression product, the transgene being integrated into a specific genomic locus defined by nucleotides 29521 to 39747 of an endogenous SEQ ID NO: 1, and the cells express the product of the transgene at a level higher than that achieved when the transgene is randomly integrated into the genome of the cells.

2. The engineered CHO cells according to claim 1, characterized in that, The genome also includes a second allele of the genomic locus, the second allele containing the endogenous nucleotide sequence of SEQ ID NO: 2 from position 29520 to 30520.

3. The engineered CHO cells of claim 1 or 2, wherein the engineered CHO cells are engineered CHO-K1 cells.

4. The engineered CHO cells of claim 1 or 2, wherein the engineered CHO cells lack expressed endogenous retroviral sequences (ERV), and / or the culture supernatant of the cells does not contain detectable viral particles.

5. The engineered CHO cells of claim 1 or 2, wherein at least one transgenic expression product is a protein of interest.

6. The engineered CHO cell of claim 1 or 2, wherein the engineered CHO cell expresses the protein of interest at least 1.5 times, 2 times, 2.5 times, 3 times or more per unit time than the amount of the protein of interest when the at least one transgene is integrated into the genome outside the locus.

7. A cell population comprising engineered CHO cells according to any one of claims 1-6, wherein at least one transgene is integrated into more than 20%, 30%, or even more than 40% of the cells in the cell population.

8. A method for producing engineered CHO cells according to any one of claims 1-6, the method comprising the following steps: (a) Transfect CHO cells using the following method: (i) at least one nuclease and / or cleavage enzyme targeting a genomic locus defined by nucleotides 29521 to 39747 of an endogenous SEQ ID NO: 1; and (ii) At least one genetically modified organism; as well as (b) Integrating the at least one transgene into the locus.

9. The method of claim 8, wherein the at least one nuclease and / or cleaving enzyme is selected from the group consisting of: RNA-guided nucleases as part of the CRISPR system, and transcription activator-like effector (TALE) nucleases or cleaving enzymes.

10. The method of claim 8, wherein the at least one transgene is provided as part of a vector.

11. The method of claim 10, wherein the vector is a plasmid or a viral vector.

12. The method of claim 8, wherein the nuclease and / or cleavage enzyme is encoded by at least one vector.

13. The method of claim 8, wherein the method further comprises providing at least one vector encoding at least one targeting element that guides the at least one nuclease and / or cleavage enzyme.

14. The method of claim 8, wherein the method further comprises regulating DNA repair pathways in the CHO cells to promote the integration of the at least one transgene into the locus.

15. The method of claim 14, wherein the DNA repair pathway is microhomology-mediated end joining (MMEJ) or homologous recombination (HR).

16. The method of claim 8, wherein the method further comprises isolating engineered CHO cells containing the at least one transgene integrated into the locus.

17. The method of claim 9, wherein the RNA-guided nuclease is a Cas9 nuclease.

18. The method of claim 9, wherein the TALE nuclease or cleavage enzyme is engineered to recognize the sequence within the locus.

19. A kit for introducing at least one transgene into CHO cells, said kit comprising: A vector encoding at least one nuclease and / or cleavage enzyme that targets a genomic locus in the CHO cells, the locus being defined by the endogenous nucleotide sequence of SEQ ID NO: 1 from position 29521 to 39747; At least one stimulator and / or inhibitor of a DNA repair pathway in a separate container, wherein the DNA repair pathway is microhomology-mediated end joining (MMEJ) or homologous recombination (HR). Instructions for use of the at least one nuclease and / or cleavage enzyme and the at least one stimulator and / or inhibitor to transfect the cells with the at least one transgene.

20. The kit of claim 19, wherein the kit further comprises at least one vector encoding at least one targeting element that guides the at least one nuclease and / or cleaving enzyme, the at least one vector being contained in a container containing a vector encoding the at least one nuclease and / or cleaving enzyme that targets the genomic locus, or being contained in a separate container.

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