Versatile HSV-1 pre-vector

Abstract

The present invention is directed to a viral expression backbone, a viral expression vector produced from the backbone, and a pharmaceutical composition thereof that are suitable for use therapeutically or for in vitro applications, and methods relating thereto. In other words, the size of the genome of the modified HSV-1 vector produced from the HSV-1 pre-vector of the invention is comparable to the size of the genome of the wild- type HSV-1 genome.

Description

[0001] TITLE: VERSATILE HSV-1 PRE- VECTOR

[0002] BACKGROUND

[0003] Herpes simplex virus (HSV) is a complex, non-integrating DNA virus capable of infecting a very wide range of human and animal cells. HSV encompasses two serotypes, herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). The genome of HSV-1 has a size of approximately 152- kilo base pairs (kbp). It contains some 90 proteinencoding genes and more than 12 microRNA. The HSV-1 genome is composed of two unique segments, UL and US, each flanked by inverted repeats that encode critical genes present in two copies.

[0004] HSV-1 -based vectors have been investigated for use as gene transfer vectors, including for possible therapeutic use in human patients. Such vectors are modified to include a transgene which can be expressed in cells or tissues. In the case of non-replicative recombinant vectors, the vector genomes are further modified to remove essential immediate- early (IE) genes such as ICP27 and ICP4, which renders the virus completely defective and incapable of expression of early (E) genes, involved in viral genome replication, and late (L) genes, necessary for progeny virion assembly. These replication-defective viruses can be grown on complementing cells that express (complement) the missing gene products and can then be used to infect non-complementing cells, where the viral genome expresses only the therapeutic transgene. However, removal of genes from the HSV genome can have negative effects on vector productivity, genomic stability and / or transgene expression.

[0005] It remains difficult and expensive to obtain large amounts of high-titer, high purity and non-pathogenic vector stocks, in spite of several important advances in this field.

[0006] Thus, there remains a need for non-replicative HSV-1 vectors that can be modified to include one or more transgenes of interest but are non-toxic to cells or tissues, and that are capable of being efficiently multiplied while maintaining the ability to efficiently express a transgene.

[0007] SUMMARY OF THE INVENTION

[0008] The present invention is directed to a viral expression backbone, a viral expression vector produced from the backbone, and a pharmaceutical composition thereof that are suitable for use therapeutically or for in vitro applications, and methods relating thereto. In one embodiment, the invention provides an HSV-1 pre-vector comprising a HSV-1 genome wherein non-essential genes, essential genes, or combinations thereof have been deleted to arrive at a genome comprising less than 130 kbp and greater than 75 kbp.

[0009] In one embodiment, the invention provides a modified HSV-1 vector comprising an HSV-1 pre-vector according to the invention wherein one or more nucleic acids and one or more st ffers, as defined herein, are introduced into the HSV-1 pre-vector. In embodiments, the one or more nucleic acids is a transgene of interest. In embodiments, the transgene is part of an expression cassette.

[0010] In one embodiment, the invention provides a kit comprising the HSV-1 pre- vector according to the invention and instructions. In embodiments, the kit further optionally comprises a plurality of first vials, each first vial independently comprising a stuffer of various lengths. In embodiments, the kit further optionally comprises a plurality of second vials, each second vial independently comprising a nucleic acid encoding a cell targeting protein.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Fig. 1A through 1J depict various embodiments of HSV-1 pre- vectors according to the invention.

[0013] Fig. 1A depicts a universal BAC-A_ldel single-copy vector backbone.

[0014] Fig. IB Universal BAC-A double-copy vector backbone.

[0015] Fig. 1C Universal BAC-B1 A-gD de-targeted vector backbone.

[0016] Fig. ID Universal A-ICPO de-targeted vector backbone.

[0017] Fig. IE Universal A-gD + A-ICPO de-targeted vector backbone.

[0018] Fig. IF Universal single-copy BAC-A_ldel +AUL39-41 vector backbone.

[0019] Fig. 1G Universal single-copy BAC-A_ldel+ AUL39-41+ AUL43-47 vector backbone.

[0020] Fig. 1H Universal backbone single-copy BAC-A_ldel+ AUL39-41+ AUL43-47 + AUS2-US6 vector backbone.

[0021] Fig. 11 Universal backbone single-copy BAC-A_ldel+ AUL39-41+ AUL43-47 + AUS7-US12 vector backbone.

[0022] Fig. 1J Universal backbone single-copy BAC-A_ldel+ AUL39-41+ AUL43-47 + AUS2-US12 vector backbone. Fig. 2A through Fig. 2F depict various embodiments of inserting stuffer into the pre- HSV-1 vectors according to the invention.

[0023] Fig. 2A. Universal BAC-A_ldel single-copy vector backbone including stuffer in A- ICP4.

[0024] Fig. 2B Universal BAC-A_ldel single-copy vector backbone including stuffer in A- ICP4 and intergenic region.

[0025] Fig. 2C Universal BAC-A_ldel single-copy vector backbone including stuffer in A- joint.

[0026] Fig. 2D Universal single-copy BAC-A_ldel + AUL39-41 + AUL43-47 + AUS2- US12 vector backbone including stuffer in intergenic region and in A-joint 1 and A-joint 4.

[0027] Fig. 2E. Universal BAC-B1 A-gD re-targeted vector backbone.

[0028] Fig. 2F Universal BAC-A_ Stuffer in Joint region and transgene in the LAT region

[0029] Fig. 3 depicts a HSV-1 pre-vector that is a backbone for a modified HSV-1 vector with optimal size, upon transfection into mammalian cell and concomitant excision of B AC sequence. Fig. 3 depicts the HSV-1 pre-vector without stuffer, size about 140,978 bp including the BAC sequence (about 131,533 bp without including the BAC sequence).

[0030] Fig. 4A to Fig. 4C depicts various embodiments of a modified HSV-1 vector.

[0031] Fig. 4A depicts an embodiment of a modified HSV-1 vector comprising a nucleic acid sequence of interest (i.e., a transgene) without stuffer and after BAC excision, final DNA size about 131,559 bp.

[0032] Fig. 4B depicts an embodiment of a modified HSV-1 vector comprising a nucleic acid sequence of interest (i.e., a transgene) with stuffer before excision of BAC module upon transfection in mammalian cell, size about 162,978 bp.

[0033] Fig. 4C depicts the modified HSV-1 vector of Fig. 4B after auto-excision of BAC module, size about 153,533 bp.

[0034] Fig. 5A to Fig. 5C depicts images showing the results of Oxford Nanopore Technology sequencing of modified HSV-1 vectors with a fragment containing stuffer.

[0035] Fig. 5 A Sequencing of DNA of Modified HSV-1 vector shown Fig. 4C) and without the stuffer Fig. 5B (Sequencing of DNA of Modified HSV-1 vector shown Fig. 4A showing amplicon population) and

[0036] Fig. 5C (Sequencing of DNA of a Modified HSV-1 vector with original size 138,012 bp showing DNA region duplication (9,000 bp)). Recombination by-products amplicons (Fig. 5B), and genome duplication (Fig. 5C) due to shorter size of the genome, have been indicated with red arrows.

[0037] Fig. 6 depicts the impact of genome stability of multiple Modified HSV-1 vectors with different sizes, over multiple passages.

[0038] DETAILED DESCRIPTION

[0039] Features may be described herein as part of the same or separate aspects or embodiments of the present invention for the purpose of clarity and a concise description. It will be appreciated by the skilled person that the scope of the invention may include embodiments having combinations of all or some of the features described herein as part of the same or separate embodiments.

[0040] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0041] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0042] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers with that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0043] As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0044] The wild-type genome of HSV-1 has a size of approximately 152-kbp and comprises some 90 protein-encoding genes and at least 12 microRNA. However, a large part of this genome encodes non-essential genes that could in principle be individually deleted without significantly perturbing virus multiplication and packaging in cultured cells. In addition, one or more essential genes can also be deleted, but in this case the virus will only multiply in the presence of a complementing system, such as a complementing cell line, that provides the proteins not expressed by the vector genome. Therefore, the genome of HSV-1 can be modified such that non-essential genes, essential genes, or combinations thereof can be deleted. Altogether, these deletions can create a vast genomic space allowing the introduction and delivery of very large foreign pieces of DNA. pre-HSV-1 vector

[0045] In one embodiment, the invention provides a mini-HSV-1 backbone (aka “mini-HSV- 1” or “a HSV-1 pre-vector”) wherein non-essential genes, essential genes, or combinations thereof have been deleted from the HSV-1 genome to arrive at a genome backbone comprising less than 130 kbp and greater than 75 kbp. HSV-1 pre- vectors embodying the invention are described with the understanding that, as "backbones," it is contemplated that one or more nucleic acids, with or without extraneous control elements, can be inserted therein.

[0046] According to the invention, the qualifier “essential” in the expressions “essential genes” or “non-essential genes”, means that the given gene is essential (or not) for achieving multiplication and packaging of the virus genome, thus generating infectious progeny virus particles. HSV-1 essential genes include ULI, UL5-UL9, UL12, UL14, UL15, UL17-UL19, UL22, UL25-UL38, UL42, UL48, UL49, UL52, UL54, US6, ICP4 (2 copies). HSV-1 non- essential genes include ICP34.5 (2 copies), ICPO (2 copies), LAT (2 copies), UL2-UL4, UL10, UL11, UL13, UL16, UL20, UL21, UL23, UL24, UL39, UL40, UL41, UL43-UL47, UL50, UL51, UL 53, UL55, UL56, US1-US5, US7-US12.

[0047] In embodiments, clusters of genes that could be deleted include, but are not limited to, genes UL2, UL3, UL4 (10.200 - 12.600); genes UL10, UL11 (23.200 - 25.200); gene UL16 (30.200 - 31.400); genes UL20, UL21 (40.800 - 43.700); genes UL23, UL24 (46.700 - 48.600); genes UL39, UL40, UL41 (86.400 -92.700); genes UL43 to UL47 (94.700 - 103.200); genes UL50, UL51 (107.700 - 109.100); genes UL55, UL56 (115.400 - 117.100); one copy of genes LAT, ICPO, UL34.5 (IRL) (118.700 - 126.100); genes US2 to US5 (134.000 - 138.200); genes US7 to US12 (139.700 - 145.600); and / or the second copy of gene ICPO when the first copy has already been removed among the LAT, ICPO, UL34.5 cluster.

[0048] In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 23 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 30 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 40 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 45 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 50 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 55 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 60 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 65 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 75 kbp have been deleted.

[0049] In some embodiments, the HSV-1 pre-vector comprises a genome wherein 25 kbp to 80 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein 30 kbp to 75 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein 35 kbp to 70 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein 40 kbp to 60 kbp have been deleted.

[0050] In embodiments the invention provides a HSV-1 pre-vector comprising a HSV-1 genome wherein non-essential genes, essential genes, or combinations thereof have been deleted to arrive at a genome backbone comprising less than 130 kbp and greater than 75 kbp.

[0051] In embodiments, the HSV-1 pre- vector further comprises a Bacterial Artificial Chromosome (BAC) sequence. As used herein, a BAC is an engineered DNA molecule used in a fashion that allows it to be propagated as a circular artificial chromosome in bacteria. BAC vectors are plasmids constructed with the replication origin of E. coli F factor, and so can be maintained in a single copy per cell. These vectors can hold DNA fragments of up to 300 kb (see e.g., Figure 3 and 4).

[0052] Although all non-essential HSV-1 genes can in principle be deleted within a BAC, which is propagated in bacteria, the collective deletion of all of them will most probably create a disabled virus, too much attenuated to be efficiently grown in mammalian cultured cells. In addition, the size of the HSV-1 pre- vector genome is itself important as a genome that is too short or too large cannot be correctly packaged, hence the need to compensate for the size.

[0053] It is important that the HSV-1 pre-vectors of the invention maintain enough of the HSV-1 genome so as to not become HSV-1 amplicons. By "Amplicon or amplicon vector" it is meant a helper-dependent vector, the genome of which lacks most or all HSV genes coding for virus proteins. The genome of amplicon vectors is a concatemeric DNA composed of multiple copies in tandem of a plasmid -known as the amplicon plasmid- that carries one origin of DNA replication and one packaging signal from HSV-1 genome. In cells expressing the full set of structural, replication and DNA packaging functions from HSV-1, resulting from the presence of an HSV-1 genome acting as helper, the amplicon plasmid is amplified by a rolling-circle mechanism into long head-to-tail concatemers that are then cleaved and packaged, up to one genome size, into HSV-1 virions (Kwong and Frenkel, 1985; Bataille and Epstein, 1997). Amplicon vectors are thus concatemeric plasmidic DNA packaged into HSV-1 particles.

[0054] Modified HSV-1 Vector

[0055] As described herein, the HSV-1 pre- vector of the invention comprises deletions to the HSV genome so that the HSV-1 pre- vector comprises a genome backbone having less than 130 kbp and greater than 75 kbp. As discussed above, these pre-vectors "backbones" of the invention are vector templates and the deletions create genomic space allowing the introduction of one or more nucleic acid sequences of interest. Introduction of the one or more nucleic acid sequences, e.g., a transgene of interest, increases the genome size (i.e., number of base pairs) in the resulting modified HSV-1 vector of the invention. Without wishing to be bound to any particular theory, it is believed that when the modified HSV-1 vector of the invention is of a size similar to the 152 kbp of the wild-type HSV-1 genome, the resulting modified HSV-1 vector more stable genomically and, thus, vector viability.

[0056] In embodiments, the invention provides a modified HSV-1 vector comprising one or more nucleic acid sequences of interest and one or more stuffers, wherein the size of the modified HSV-1 vector genome is about 138 kbp to about 168 kbp.

[0057] In embodiments, the invention provides a modified HSV-1 vector comprising one or more nucleic acid sequences of interest and one or more stuffers, wherein the size of the modified HSV-1 vector genome is about 142 kbp to about 160 kbp.

[0058] In any embodiment disclosed herein, at least one of the one or more nucleic acid sequences of interest encodes a protein of interest.

[0059] In any embodiment disclosed herein, the one or more nucleic acid sequences of interest comprises one or more transgenes of interest, one or more nucleic acids encoding a cell targeting protein, or combinations thereof. In any embodiment disclosed herein, the one or more nucleic acid sequences comprises one or more transgenes of interest. For clarity, the term “one or more nucleic acid sequences of interest” does not include the one or more stuffers as described herein. In any embodiment disclosed herein, the one or more nucleic acid sequences of interest can be introduced into the HSV-1 pre- vector backbone by site specific recombination (SSR), by homologous recombination (HR), or En passant mutagenesis technique. In any embodiment disclosed herein, the one or more nucleic acid sequences of interest are introduced by homologous recombination or En passant mutagenesis technique.

[0060] In an embodiment, the modified HSV-1 vector comprises one or more transgenes and one or more stuffers, wherein the modified HSV-1 vector is capable of persistent expression of the one or more transgenes, such as a therapeutic transgene.

[0061] In any embodiment herein, the size of the modified HSV-1 genome after addition of the one or more stuffers and one or more nucleic acids of interest is between about 138 kbp and 168 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 142 kbp and 164 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 142 kbp and 155 kbp. In a preferred embodiment herein, the size of the modified HSV-1 genome is between about 142 kbp and 153 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 147 kbp and 159 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 147 kbp and 156 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 150 kbp and 156 kbp. In any embodiment herein, the size of the modified HSV-1 genome is about 153 kbp.

[0062] In any embodiment herein, the size of the modified HSV-1 genome after addition of the one or more stuffers and one or more nucleic acids of interest is between about 142 kbp and 160 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 142 kbp and 158 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 142 kbp and 155 kbp. In a preferred embodiment herein, the size of the modified HSV-1 genome is between about 142 kbp and 153 kbp.

[0063] In any embodiment herein, the size of the modified HSV-1 genome is between about 143 kbp and 160 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 143 kbp and 158 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 143 kbp and 155 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 143 kbp and 153 kbp.

[0064] In any embodiment herein, the size of the modified HSV-1 genome is between about 145 kbp and 160 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 145 kbp and 158 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 145 kbp and 155 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 145 kbp and 153 kbp.

[0065] In any embodiment herein, the size of the modified HSV-1 genome is between about 147 kbp and 160 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 147 kbp and 158 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 147 kbp and 155 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 147 kbp and 153 kbp.

[0066] In any embodiment herein, the size of the modified HSV-1 genome is between about

[0067] 149 kbp and 160 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 149 kbp and 158 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 149 kbp and 155 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 149 kbp and 153 kbp.

[0068] In any embodiment herein, the size of the modified HSV-1 genome is between about

[0069] 150 kbp and 155 kbp. In any embodiment herein, the size of the modified HSV-1 genome is between about 150 kbp and 153 kbp.

[0070] As used herein, the term “stuffer” refers to any random non-coding DNA sequence, and / or one or more genes of interest, or combinations thereof. The primary purpose of the stuffer is to reintroduce a nucleic acid of an appropriate size (in base pairs) so that the size of the modified HSV-1 vector, based on the starting size of the HSV-1 pre- vector, is at or close to the size of the wild type HSV-1 genome, i.e., about 152 kbp, after all other nucleic acids of interest (e.g., one or more transgenes and / or one or more exogenous nucleic acids and / or one or more endogenous nucleic acids) has been added to the HSV-1 pre- vector. Considerations for the stuffer include the nature of the stuffer, the size of the stuffer, and the placement of the stuffer in the genome of the modified HSV-1 vector of the invention.

[0071] Therefore, the design of the stuffer is determined last after all other design choices for the modified HSV-1 vector have been made. For example, if the HSV-1 pre-vector of the invention comprises 105 kilo base pairs (kbps) and a transgene, as describe herein, comprising 5 kbps is introduced, then one or more stuffers comprising about 33 kbps to about 58 kbps, in total, is to be introduced into the modified HSV-1 vector. Alternatively, in this scenario, one or more stuffers comprising about 36 kbps to about 50 kbps, in total, is introduced. Alternatively, in this scenario, one or more stuffers comprising about 39 kbps to about 47 kbps, in total, is introduced. Alternatively, in this scenario, one or more stuffers comprising about 42 kbps, in total, is introduced. In embodiments, the one or more stuffers comprise a non-coding DNA sequence. For example, in one embodiment a scrambled sequence from the HSV-1 genome (e.g., scrambled HSV-1 DNA) can be used. A scrambled sequence of HSV-1 DNA can comprise sequences of no more than 20 nucleotides each but representative of the whole genome to keep similar the GC content and the “natural style”, e.g., “natural behavior”, of HSV-1 DNA.

[0072] In embodiments, the one or more stuffer sequences have a GC content ranging from 60% to 75%. In embodiments, the stuffer sequence has a GC content ranging from 65% to 69%. In preferred embodiments, the stuffer sequence has a GC content ranging from 67% to 68%.

[0073] Sequences of no more than 20 nucleotides can be repeated until the one or more stuffers are at the desired size. These sequences can be taken from non-coding DNA to avoid introducing transcriptionally significative signals. In addition, sequences such as Kozak sequence, initiation codons, repeated sequences, potential miRNA clusters, and long palindromes should not be included. In addition, sequences that are present in the human genome or in the manufacturing cell line genome should not be included to avoid vector genome recombination. Stop codons can be regularly introduced to avoid any long open reading frame. In general, the stuffer of random DNA should avoid having any biological sense, to restrict its usefulness exclusively to the increase in size.

[0074] In some embodiments, the one or more stuffers can be a scrambled nucleotide sequence of genes deleted from the HSV-1 genome.

[0075] In embodiments, the one or more stuffers can originate from cellular DNA introns modified to avoid vector genome recombination.

[0076] In embodiments, the stuffer can be divided in two or more fragments, inserted in different positions in the HSV-1 pre- vector genome. In preferred embodiments, the two or more stuffer fragments should not present homology sequences to avoid vector genome internal recombination.

[0077] In embodiments, the primary purpose of the one or more stuffers can be achieved by any random non-coding DNA sequence and / or one or more genes of interest, or combinations thereof. That is, the role of the “stuffer” to bring the size of the modified HSV-1 vector genome back up to about 152 kb, can be accomplished by the use of any random non-coding DNA sequence alone. Alternatively, in some embodiments, the role of the stuffer can be accomplished by the use of one or more HSV-1 essential genes, one or more HSV-1 non- essential genes, one or more exogenous genes of interest (as defined herein) alone or combinations thereof with or without the use of one or more random non-coding DNA sequence.

[0078] The term "transgene" refers to a particular nucleic acid sequence encoding for a coding or non-coding RNA and / or a polypeptide or a portion of a polypeptide to be expressed in a cell into which the nucleic acid sequence is introduced. The term "transgene" includes (1) a nucleic acid sequence that is not naturally found in the cell (i.e., a heterologous nucleic acid sequence); (2) a nucleic acid sequence that is a mutant form of a nucleic acid sequence naturally found in the cell into which it has been introduced; (3) a nucleic acid sequence that serves to add additional copies of the same (i.e., homologous) or a similar nucleic acid sequence naturally occurring in the cell into which it has been introduced; or (4) a silent naturally occurring or homologous nucleic acid sequence whose expression is induced in the cell into which it has been introduced. By "mutant form" is meant a nucleic acid sequence that contains one or more nucleotides that are different from the wild-type or naturally occurring sequence, i.e., the mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and / or insertions. In some cases, the transgene may also include a sequence encoding a leader peptide or signal sequence such that the transgene product will be secreted from the cell, or the transgene may include both a leader peptide or signal sequence plus a membrane anchor peptide, or even be a fusion protein between two naturally occurring proteins or part of them, such that the transgene will remain anchored to cell membranes, or a sequence that allows the protein to accumulate in a specific region of the cell, such as a nuclear localizing signal.

[0079] Non-limiting examples of transcribed sequences that can be present in a transgene within the inventive vector include, but are not limited to, a sequence expressing a long transgene, a sequence expressing a transgene able to generate different splice variants, a sequence expressing two or more transgenes, either as a single transcription unit or in separated transcription units, a sequence expressing a transgene under the control of very long upstream and / or downstream regulatory sequences, a sequences expressing short regulatory RNAs (miRNA, siRNA, etc.), a sequences conferring the ability to be replicated and correctly segregated in dividing cells, or combinations of the above sequences.

[0080] As noted above, the inventive vector can include at least one transgene inserted in operable connection with one or more LTEs and / or DNA insulator sequences within the HSV-1 pre- vector. By "operably connected," it is to be understood that the one or more LTEs and / or DNA insulator sequences permit the transgene to be expressed in a cellular environment in which genetic elements (i.e., "genes") otherwise present within the HSV genome are transcriptionally silent.

[0081] Within the transgene(s) inserted into the inventive vector, there are at least a promoter sequence and a transcribed sequence such that the transcribed sequence(s) is controlled by the promoter. A “promoter”, as used herein, is a DNA regulatory region capable of binding RNA polymerase in a mammalian cell and initiating transcription of an operably linked downstream (3' direction) sequence. For purposes of the present invention, a promoter sequence includes at least the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background. Within the promoter sequence is a transcription initiation site, as well as RNA polymerase binding domains. Eukaryotic promoters will often, but not always, contain "TATA" boxes and other DNA motifs, such as "CAT" or "SP1" boxes.

[0082] The promoter within the transgene can be any promoter desired to control / regulate the expression of the transcribed sequence(s). For example, the promoter can be a cell-specific or tissue-specific promoter (e.g., EOS, OCT4, Nanog (for ESC / iPSC), SOX2 (for neural stem cells), aMHC, Brachyury, Tau, GFAP, NSE, Synapsin I (for neurons), Apo A-I, Albumin, ApoE (for liver), MCK, SMC a-Actin, Myosin heavy chain, Myosin light chain (for muscle), etc.), such as a promoter that specifically or preferentially control expression of genes in a defined cell type (e.g., within a liver cell, lung cell, epithelial cell, cardiac cell, neural cell, skeletal muscle cell, embryonic, induced pluripotent, or other stem cell, cancer cell, etc.).

[0083] In embodiments, promoters for use in sensory neurons include promoters of genes coding for sensory neuroreceptors such as Transient Receptor Potential Vanilloid 1 (TRPV1) or Transient Receptor Potential cation channel subfamily M member 8 (TRPM8), or from promoters of genes coding for sensory neuromodulators or sensory neurotransmitters, such as the promoters of Substance P, PACAP, Calcitonin Gene Related Peptide (CGRP). In embodiments, promoter of genes coding for sensory neuroreceptors according to the invention is a promoter of the TRP gene family, more preferentially the promoter TRPV 1 or TRPM8. In embodiments, promoters of genes coding for sensory neuromodulators or sensory neurotransmitters according to the invention is the CGRP, or the promoter of genes involved in neurite outgrowth and stress response in sensory neurons, preferably the promoter of the gene encoding advillin (ADVL). In other embodiments, the promoter within a transgene inserted into the inventive vector can be an inducible promoter.

[0084] In any embodiment disclosed herein, the one or more transgenes can be part of one or more expression cassettes. The term "expression cassette" as used herein refers to any nucleic acid sequence containing a promoter and a downstream coding sequence or transgene, which expression is driven by said promoter, which is followed by a polyadenylation signal.

[0085] In embodiments, the promoter within a transgene expression cassette inserted into the inventive vector can be a constitutive mammalian promoter, such as are known in the art (e.g., EFla, UbC, P-actin, PGK, and the like).

[0086] In addition to the promoter(s) and coding sequence(s), the transgene(s) inserted into the genome of the inventive vector also can comprise additional regulatory element(s). For example, the transgene(s) can include one or more sites for binding of microRNA. The presence of such sites facilitates down-regulation of the transgene expression in certain cell types. Thus, for example, a vector comprising a transgene desired to be expressed specifically in a cancer or tumor cell (which may be toxic to many cell types) can comprise binding sites for microRNAs of "normal" (i.e., non-malignant) cells, so that the expression of the transgene is suppressed in non-malignant cells.

[0087] In embodiments, transgene(s) within the inventive vector can be monocistronic (i.e., encoding a single mRNA, resulting in a single protein or polypeptide) or polycistronic (i.e., encoding multiple mRNA, resulting in more than one protein or polypeptide) or can express a single or multiple mRNAs that encode self-cleavable polyproteins. In embodiments, all or part of the transcribed portion of the transgene also can encode non-translated RNA, such as siRNA or miRNA. In embodiments, the inventive vector can comprise multiple separate monocistronic or polycistronic transgene units, each with its own respective promoter, translated sequence(s) or non-translated RNA sequence(s), and other regulatory elements.

[0088] In any embodiment disclosed herein, the one or more nucleic acid sequences of interest can be introduced in various locations such as in a LAT region, in one or more intergenic regions, in one or more genic regions, or combinations thereof.

[0089] A LAT (Latency Associated Transcripts) locus or region is a repeated locus that is contained in the inverted repeated sequences known as b and b' of the virus genome. The b and b' sequences of the virus genome are also known as TRL (Terminal Repeat Long) and IRL (Internal Repeat Long), respectively. In embodiments, the pre-HSV-1 vector genome contains both LAT regions, one in the TRL and the other in the IRL. In embodiments one of the LAT regions, either in the TRL or in the IRL has been deleted. In embodiments, when the pre-HSV-1 vector genome contains the two LAT loci, foreign DNA can be introduced into both loci, in the TRL region and in the IRL region as shown in figure IB. In embodiments, when the LAT locus in the IRL region is deleted, foreign DNA can be introduced into the LAT locus in the TRL region only. In embodiments, when the LAT locus in the TRL region is deleted, foreign DNA can be introduced into the LAT locus in the IRL region only.

[0090] The LAT locus includes an upstream DNA insulator (INS) sequence, the Latency Associated Promoter (LAP), a region conferring Long-Term Expression (LTE) and a downstream DNA insulator (INS). In embodiments, foreign DNA is introduced either between the Latency Associated Promoter (LAP) and the Long-Term Expression (LTE) region (as shown in Figure 4), or between the LTE region and the DNA insulator (INS) sequence present downstream of the LTE (as shown in Figure 1A and IB).

[0091] Importantly, the LAT locus contains both the LTE and the DNA insulator sequences (INS) that confer long-term expression to any foreign DNA, e.g., a therapeutic transgene, introduced into this site. In embodiments, the foreign DNA will be localized between the long-term expression (LTE) and the downstream DNA insulating (INS) motifs.

[0092] By "long-term expression sequence" or "long-term expression element (LTE)" it is meant a nucleotide sequence that when operably linked to a foreign DNA of interest allows for sustained expression of a gene product for more than 15 to 45 days or 30 to 45 days, or from 45 to 90 days, or from 90 to 365 days, or 365 days to several years or even during the life of the patient. Long-term expression (LTE) sequences were identified in HSV-1 as a region of the latency- associated transcripts (LAT), which originate from the LAT-associated promoter (LAP). This LTE is located downstream of the LAT transcription start site. Preferably, the LTE is comprised between about 1.5 kb to about 3 kb downstream of the LAT transcription start site. Additionally, DNA insulators also contribute to provide long-term expression. Without wishing to be bound to any particular theory, DNA insulators may inhibit epigenetic silencing. Sequences conferring long-term expression (both the LTE and the DNA insulator sequences) can be placed either upstream and / or downstream the foreign DNA.

[0093] Those of skill in the art will recognize that other LTE-like sequences, as well as other DNA insulator sequences, have been described and are continually being discovered. All such LTE-like sequences and DNA insulator sequences are encompassed by the present invention.

[0094] As used herein, the term, one or more exogenous genes of interest can include, but are not limited to, a reporter gene (e.g., GFP, RFP, luciferase, or fused protein, etc.) driven by a transient promoter serving as internal expression control or for biodistribution studies; recombinases driven by an inducible promoter to allow in vivo modifying cellular or viral genes; antibiotic resistance genes such as chloramphenicol; elements of the Tetracycline inducible system (TRE); any foreign DNA encoding a gene of interest, or combinations thereof. The use of reporter genes such as, but not limited to, cherry, RFP, GFP, or CFP, can facilitate the identification of the recombined genome, to assess the stability of the stuffer and to score both infectious particles (PFU) and transducing units (TU).

[0095] In embodiments, the modified HSV-1 vector of the invention comprises two or more nucleic acid sequences of interest, which can be the same or different, and can be introduced in one or more intergenic regions (IR) of the modified HSV-1 vector. As used herein, an intergenic region generally refers to the space between the poly(A) signals of two genes with convergent orientation. Several IRs have been described in the virus genome that allow the introduction of long foreign DNA pieces without disturbing virus multiplication of expression of neighboring genes, for example the IR localized between genes UE3 and UE4, genes UE7 and U8, genes UE26 and UE27, or genes UE35 and UE36, amongst others. In embodiments, one of the nucleic acid sequences is introduced into a EAT region.

[0096] In embodiments, the modified HSV-1 vector of the invention further comprises a third nucleic acid sequence of interest, which can be the same or different from the first and / or second nucleic acid sequences of interest and can be introduced in an intergenic region (IR) or in a genic region. In embodiments, the third nucleic acid sequence of interest is introduced in an intergenic region (IR) different from the IR for the first and second nucleic acid sequences of interest. In embodiments, the third nucleic acid sequence of interest is introduced in a genic region.

[0097] In embodiments, the third nucleic acid sequence of interest comprises exogenous or endogenous DNA or combinations thereof into the modified HSV-1 vector of the invention. In embodiments, the third nucleic acid sequence of interest provides exogenous DNA into the modified HSV-1 vector. In embodiments, the third nucleic acid sequence provides endogenous DNA into the modified HSV-1 vector. In embodiments, the third nucleic acid sequence of interest provides combinations of exogenous and endogenous DNA into the modified HSV-1 vector.

[0098] In embodiments, the introduction of third nucleic acid sequence of interest satisfies two important roles. The first is to introduce DNA of interest into the modified HSV-1 vector. The second purpose is to bring the size of a modified HSV-1 vector produced from the HSV-1 pre-vector to a final size of about 152 kb, or between 142kb and 155kb. In other words, the size of the genome of the modified HSV-1 vector produced from the HSV-1 pre- vector of the invention is comparable to the size of the genome of the wild- type HSV-1 genome.

[0099] In some embodiments, the third nucleic acid sequence to be introduced is selected from one or more HSV-1 essential genes, one or more HSV-1 non-essential genes, one or more exogenous genes of interest (as defined herein) or combinations thereof.

[0100] In some embodiments, the third nucleic acid sequence is one or more HSV-1 essential genes. In some embodiments, the third nucleic acid sequence is a combination of a stuff er as defined herein and one or more HSV-1 essential genes.

[0101] In some embodiments, the third nucleic acid sequence is one or more HSV-1 non- essential genes. In some embodiments, the third nucleic acid sequence is a combination of the stuffer and one or more HSV-1 non-essential genes. In some embodiments, the third nucleic acid sequence is a combination of stuffer, one or more HSV-1 essential genes, and one or more HSV-1 non-essential genes. In some embodiments, the third nucleic acid sequence is a combination of one or more HSV-1 essential genes and one or more HSV-1 non-essential genes.

[0102] In some embodiments, the third nucleic acid sequence is one or more exogenous genes of interest. In some embodiments, the third nucleic acid sequence is a combination of stuffer and one or more exogenous genes of interest. In some embodiments, the third nucleic acid sequence is a combination of stuffer, one or more HSV-1 essential genes, and one or more exogenous genes of interest. In some embodiments, the third nucleic acid sequence is a combination of stuffer, one or more HSV-1 non-essential genes, and one or more exogenous genes of interest. In some embodiments, the third nucleic acid sequence is a combination of one or more HSV-1 essential genes, one or more HSV-1 non-essential genes, and one or more exogenous genes of interest. In some embodiments, the third nucleic acid sequence is a combination of one or more HSV-1 essential genes and one or more exogenous genes of interest. In some embodiments, the third nucleic acid sequence nucleic acid sequence is a combination of one or more HSV-1 non-essential genes and one or more exogenous genes of interest.

[0103] In embodiments, when introduced into a genic region, the third nucleic acid sequence can be introduced in the region comprising the HSV essential gene encoding glycoprotein D (gD) thereby resulting in an inactivating deletion of gD. In some embodiments, the inactivating deletion of gD comprises a deletion of the entire coding sequence of gD. Deletion of gD and introduction of the third nucleic acid sequence in its place allows for the replacement of the gD sequence with another cell targeting sequence. In some embodiments, the original gD sequence or a modified version thereof can be reintroduced by the third nucleic acid sequence.

[0104] In some embodiments, the inactivating deletion of gD comprises a deletion of a part of the coding sequence of gD. For example, the inactivating deletion can comprise a deletion of the amino acids 6-38 in gD, which is a region primarily responsible for cell targeting. Introduction of the third nucleic acid sequence in this region allows for the replacement of the gD cell targeting sequence with another cell targeting sequence. For example, the gD cell targeting sequence can be replaced with scFv (single chain antibody) to HER2. In some embodiments, the original gD cell targeting sequence or a modified version thereof can be reintroduced by the third nucleic acid sequence.

[0105] In some embodiments, the modified HSV-1 vector of the invention comprises at least a fourth nucleic acid sequence; wherein the fourth nucleic acid sequence comprises one or more HSV-1 essential genes, one or more HSV-1 non-essential genes, one or more exogenous genes of interest or combinations thereof.

[0106] In some embodiments, the modified HSV-1 vector of the invention comprises at least a fourth nucleic acid sequence; wherein the fifth nucleic acid sequence comprises one or more HSV-1 essential genes, one or more HSV-1 non-essential genes, one or more exogenous genes of interest or combinations thereof.

[0107] In some embodiments, the modified HSV-1 vector comprises one or more nucleic acid sequences comprising a DNA sequence, for example an endogenous and / or foreign DNA sequence encoding a cell targeting protein. The cell targeting protein can retarget virus entry into any tissue or cell type of interest. Cell targeting genes for insertion in the modified HSV-1 vector of the invention can include, but are not limited to, HER-2, IL13a2 or modified versions thereof. Examples of modified gD can be found, for example, in EP3469071, which is incorporated herein by reference.

[0108] In any embodiments herein, the size of the one or more stuffers will be determined based on the size of the other nucleic acid sequences introduced (e.g., one or more transgenes and / or one or more nucleic acids encoding cell targeting proteins) into the modified HSV-1 vector.

[0109] For example, if the HSV-1 pre-vector of the invention comprises 105 kilo base pairs (kbps) and a transgene comprising 5 kbps and a nucleic acid encoding a cell targeting sequence, as describe herein, comprising 3 kbps is introduced, then one or more stuffers comprising about 25 kbps to about 55 kbps, in total, is introduced. Alternatively, in this scenario, one or more stuffers comprising about 33 kbps to about 47 kbps, in total, is introduced. Alternatively, in this scenario, one or more st ffers comprising about 36 kbps to about 44 kbps, in total, is introduced. Alternatively, in this scenario, one or more stuffers comprising about 40 kbps is introduced.

[0110] For example, if the HSV-1 pre-vector of the invention comprises 105 kilo base pairs (kbps) and a transgene comprising 5 kbps and a nucleic acid encoding a cell targeting sequence, as describe herein, comprising 3 kbps is introduced, then one or more stuffers comprising about 30 kbps to about 50 kbps, in total, is introduced. Alternatively, in this scenario, one or more stuffers comprising about 32 kbps to about 45 kbps, in total, is introduced. Alternatively, in this scenario, one or more stuffers comprising about 34 kbps to about 42 kbps, in total, is introduced. Alternatively, in this scenario, one or more stuffers comprising about 39 kbps is introduced.

[0111] In any embodiment herein, the stuffer can be introduced into the modified HSV-1 vector of the invention as a single, long nucleic acid sequence. In embodiments, the stuffer can be introduced into the modified HSV-1 vector of the invention as two or more stuffers of the same or different lengths.

[0112] In embodiments, the stuffer can be introduced as several, smaller stuffers in different intergenic regions of the modified HSV-1 genome. In embodiments, the smaller stuffer can be the same or different lengths.

[0113] It is important to note that a BAC sequence, when present in the HSV-1 pre-vector, can be removed when preparing the modified HSV-1 vector of the invention. As such, when removed in the final modified HSV-1 vector, the size of the BAC sequence is not considered when determining the size of the one or more stuffers so that the size of the modified HSV-1 vector is about 152 kbp.

[0114] The production of any modified HSV-1 vector prepared from the HSV-1 pre- vector will thus require the concurrent construction of a cell line simultaneously complementing the deleted essential genes and optionally one or more of these non-essential genes.

[0115] Therefore, to facilitate growing, producing, and propagating the modified HSV-1 vectors of the invention and producing stocks thereof, an aspect of the invention provides a complementing cell line, which complements the genes deleted from the HSV genome. Thus, a preferred complementing cell according to the present invention is derived from a cell type that naturally complements the HSV genes that have been deleted. Such cells can be engineered to express such genes by methods known in the art (e.g., by introducing expression cassettes within the cells so that they express the genes from genetic constructs other than the HSV genome, such as the cellular chromosomes). Additionally, the complementing cell line can be engineered to express a gene encoding a selectable marker, such as markers typically employed in engineering packaging cells or cells expressing any other foreign gene. Suitable selectable genes include those conferring resistance to neomycin / G418, hygromycin, blasticidin, puromycin, zeocin, and the like.

[0116] It will be understood that methods for engineering a source cell type (e.g., Vero cells) to contain expression constructs encoding the deleted HSV proteins, as well as other proteins (such as the recombinase and / or the selectable gene product) are known to persons of ordinary skill. For example, the gene of interest with a selectable marker can be subcloned into lentiviral vectors, the source cell infected with the lentiviral vectors, selected for expression of the marker (e.g., blasticidin resistance), and then expression of the gene of interest confirmed.

[0117] The inventive complementing cell can be propagated and cloned. Thus, the invention provides a clonal population, i.e., a cell line, comprising or consisting of or essentially of the complementing cell line as described herein.

[0118] Using the inventive complementing cells of the invention, the inventive modified HSV-1 vector can be propagated. Accordingly, the invention provides a method of propagating the modified HSV-1 vector of the present invention. In accordance with the inventive method, the complementing cell line is infected with the modified HSV-1 vector and then cultured until plaques form. The viral population is amplified by repeated transfer of infectious particles to increasingly large, fresh populations of the complementing cells. For these repeated transfers, multiplicity of infection (MOI) can be between about 0.001 pfu / cell and about 0.03 pfu / cell. Ultimately, the inventive vectors (as packaged viruses) are purified from the cells at 90% cytopathic effect.

[0119] Generally, the inventive modified HSV-1 vector is most useful when enough of the virus can be delivered to a cell population to ensure that the cells are confronted with a suitable number of viruses. Thus, the present invention provides a stock, preferably a homogeneous stock, comprising the inventive modified HSV-1 vector. The preparation and analysis of HSV stocks is well known in the art. For example, a viral stock can be manufactured in roller bottles containing cells infected with the HSV-1 vector. The viral stock can then be purified on a continuous gradient, and aliquoted and stored until needed. Viral stocks vary considerably in titer, depending largely on viral genotype and the protocol and cell lines used to prepare them. Preferably, such a stock has a viral titer of about 106pfu / ml or even more preferably about 107pfu / ml (or at least about such values). In still more preferred embodiments, the titer can be about 108pfu / ml, or about 109pfu / ml (or at least about such values), and high titer stocks of about IO10pfu / ml or about 1011pfu / ml or even about 1012pfu / ml (or at least about such values) are most preferred. Thus, the titer of the HSV-1 vector stock according to the present invention can vary from about 106pfu / ml to about 1012pfu / ml (preferably between about 109to about 101 1pfu / ml).

[0120] The invention additionally provides a composition comprising the HSV-1 pre- vector and / or the modified HSV-1 vector of the invention and a physiologically-acceptable carrier. The carrier of the composition can be any suitable carrier for the vector. The carrier desirably is a pharmaceutically acceptable (e.g., a physiologically or pharmacologically acceptable) carrier (e.g., excipient or diluent). Pharmaceutically acceptable carriers are well known and are readily available. The choice of carrier will be determined, at least in part, by the particular vector and the particular method used to administer the composition. The composition can further comprise any other suitable components, especially for enhancing the stability of the composition and / or its end-use. Accordingly, there is a wide variety of suitable formulations of the composition of the invention. The following formulations and methods are merely exemplary and are in no way limiting.

[0121] Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unitdose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of a sterile liquid excipient, for example, water, for injections, immediately prior to use.

[0122] Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0123] In addition, the composition can comprise additional therapeutic or biologically- active agents. For example, therapeutic factors useful in the treatment of a particular indication can be present. Factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the vector and physiological distress. Immune system suppressors can be administered with the composition method to reduce any immune response to the vector itself or associated with a disorder. Alternatively, immune enhancers can be included in the composition to up-regulate the body's natural defenses against disease. Antibiotics, i.e., microbicides and fungicides, can be present to reduce the risk of infection associated with gene transfer procedures and other disorders.

[0124] Using the inventive modified HSV-1 vector (and stocks and compositions comprising the vector), the invention provides a method of expressing a transgene within a nucleated cell, especially a non-complementing cell. In accordance with the method, the inventive vector is exposed to the cell under conditions suitable for the vector to infect the cell. Once the cell is infected, the transgene will be transcribed (expressed) within the cell, provided the promoter within the transgene is one which is active in the cell and that the transgene is not suppressed by another regulatory mechanism (e.g., the microRNAs discussed herein). In other words, the inventive vectors serve as gene transfer and expression vectors within mammalian cells.

[0125] The invention can be employed to express transgene(s) within cells either in vivo or in vitro, as desired. For use in vivo, the cell can be any type of desired cell, such as exocrine secretory cells (e.g., glandular cells, such as salivary gland cells, mammary gland cells, sweat gland cells, digestive gland cells, etc.), hormone secreting gland cells (e.g., pituitary cells, thyroid cells, parathyroid cells, adrenal cells, etc.), ectoderm-derived cells (e.g., keratinizing epithelial cells (e.g., making up the skin and hair), wet stratified barrier epithelial cells (e.g., of the cornea, tongue, oral cavity, gastrointestinal tract, urethra, vagina, etc.), cells of the nervous system (e.g., peripheral and central neurons, glia, etc.)), mesoderm- derived cells, cells of many internal organs (such as kidney, liver, pancreas, heart, lung) bone marrow cells, and cancerous cells either within tumors or otherwise. Preferred, and non- limiting examples of cells suitable for infection by the inventive vectors include liver cells, lung cells, epithelial cells, cardiac cells, adipose cells, muscle cells, stem cells, and cancer cells.

[0126] When used in vivo, the inventive method can treat a disease or a condition within a subject, when the transgene within the vector encodes one or more prophylactically- or therapeutic ally- active proteins, polypeptides, or other factor (e.g., non-coding RNA (ncRNA) such as siRNA or miRNA). Thus, the invention provides a method of treating a disease or condition in a subject, comprising administering the vector of the present invention to the subject, in an amount and at a location sufficient to infect cells of the subject such that the transgene is expressed within the cells of the subject, and wherein the transgene encodes one or more prophylactically or therapeutically active proteins, polypeptides or ncRNA. For example, the disease or condition can be a type of cancer, in which the transgene can encode an agent that enhances tumor killing activity (such as TRAIL or tumor necrosis factor (TNF)). In other embodiments, the inventive method can be used in vitro to cause expression of the transgene within cells in culture. Again, any type of cells can be infected in vitro with the inventive method, such as stem cells and fibroblasts, such as a human dermal fibroblast (HDF) or a human lung fibroblast (HLF). Other preferred types of cells for use in vitro include keratinocytes, peripheral blood mononuclear cells, hematopoietic stem cells (CD34+), or mesenchymal stem / progenitor cells. In one embodiment, the transgene(s) encode one or more factors that can affect the differentiation of the cell.

[0127] In embodiments, infecting a cell in vivo or in vitro with the vector, composition, or stock of the invention, the cell can be any mammalian nucleated cell for which it is desired to express the transgene. Thus, the vector can be employed to infect cells of many mammalian species. It is believed that the inventive method can be applied in veterinary therapies for breeding, such as to express exogenous genes or supplement for deficient genes in animals such as cattle, horses, sheep, goats, swine, and the like. Similarly, the inventive method can be employed in a veterinary context for companion animals, such as cats, dogs, and the like.

[0128] The modified HSV-1 vectors of the invention can be used in vivo in humans as well, to provide for the expression of a prophylactic ally- or therapeutically-active agent, or factor, in a medical setting. The factor (supplied by expression of one or more of the transgenes within the inventive vectors) can be exogenous, or one that complements a genetic deficiency.

[0129] In embodiments, the invention provides a kit comprising the HSV-1 pre- vector according to the invention and instructions. In embodiments, the kit optionally further comprises a plurality of first vials, each first vial independently comprising a stuffer of the same or various lengths. In embodiments, the kit optionally further comprises a plurality of second vials, each second vial independently comprising the same or different nucleic acid encoding a cell targeting protein.

[0130] In any of the embodiments of the invention, in any of the HSV-1 pre-vectors or the modified HSV-1 vectors of the invention, the HSV-1 sub-species can be replaced with HSV- 2.

[0131] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0132] Example 1

[0133] In the preparation of a pre-HSV-1 vector according to the invention, clusters of genes that could potentially be deleted include but are not limited to: 1. Genes UL2, UL3, UL4 (10.200 - 12.600): 2.400 nt;

[0134] 2. Genes UL10, ULI 1 (23.200 - 25.200): 2.000 nt;

[0135] 3. Gene UL16 (30.200 - 31.400): 1.200 nt;

[0136] 4. Genes UL20, UL21 (40.800 - 43.700): 2.900 nt;

[0137] 5. Genes UL23, UL24 (46.700 - 48.600): 1.900 nt;

[0138] 6. Genes UL39, UL40, UL41 (86.400 -92.700): 6.300 nt;

[0139] 7. Genes UL43 to UL47 (94.700 - 103.200): 8.500 nt;

[0140] 8. Genes UL50, UL51 (107.700 - 109.100): 1.400 nt;

[0141] 9. Genes UL55, UL56 (115.400 - 117.100): 1.700 nt;

[0142] 10. One copy of genes LAT, ICP0, UL34.5 (IRL): (118.700 - 126.100): 7.400 nt;

[0143] 11. Genes US2 to US5 (134.000 - 138.200): 4.200 nt; and / or

[0144] 12. Genes US7 to US12 (139.700 - 145.600): 5.900 nt.

[0145] 45,800 nt of non-essential DNA could potentially be deleted in addition of 2 copies of ICP4 (127,200-131,300: 4,100 nt) and the single ICP27 copy (113,800-115,300: l,500nt) (so 9.7 overall). Including those 2 genes, total deletion in the backbone could be 9.7+45.8=55.5kbp. These numbers are approximated, and estimation is probably more or less 3kbp. As an intermediate level of deletion, a less defective backbone could have deletions limited to clusters number 6, 7, 10, 11 and 12 (described above). In this case, the total deletion would be of 43.5kbp.

[0146] The deletions can be introduced on a HSV-1 genome derived from the wild-type genome of HSV-1 F strain that contains a BAC module (pBeloBACl 1) that has been inserted in the intergenic region between genes UL3 and UL4 (BAC-HSV-1).

[0147] Before starting the deletion of non-essential genes, the BAC-HSV-1 can be deleted for two essential genes: ICP27 and ICP4 (both copies). Promoters of both genes ICP22 / 47 can be slightly modified by deleting the TAATGARAT box. This is therefore a non- replicative (NR) vector genome that can be grown in Vero7b (or other complementing) cells. In addition, a transgene (e.g., BoNT-F or Luc, or other) can be introduced in the LAT region. Single large deletion from ICP27 encoding gene and including the entire joint region that comprises one copy of ICP4 combined with deletion of the second copy of ICP4 results in an HSV-1 pre-vector (Fig.lA) is of a size 128kb without the BAC module.

[0148] Additions and / or deletions can be introduced by site specific recombination (SSR), by homologous recombination (HR), or by Crisper Cas. A stuff er as described herein can be introduced at the same place of the deleted genes (including ICP4 and ICP27 deletions). The deleted B AC-HSV 1 are then transfected into 7b cells to isolate, clone, and produce, the corresponding modified HSV-1 vectors.

[0149] Example 2

[0150] Two parallel strategies will be developed for the introduction of stuffer into the HSV- 1 vector. A first strategy is designed to introduce stuffers of different lengths in the same unique site and a second strategy to introduce small stuffers at different sites.

[0151] A HSV-1 vector (hereafter called BAC-3) expressing firefly luciferase (2-kbp) and still containing the BAC module (7.5-kbp) located in the intergenic region between UL3 and UL4 will be used. This BAC-3 has long deletions (about 22-kbp), a first one going from the beginning of UL54 (ICP27) to the internal oriS (deletion of UL54, 55, 56, LAT, ICPO, ICP34.5, pac, and ICP4), and a second one spanning the second copy of ICP4. Since this BAC-3 contains the transgene (2-kbp) and the BAC module (7.5-kbp), it makes a total deletion of some 13-kbp. After deleting the BAC module, the theoretical size of said BAC-3 at its starting point is about 130-kbp (20% shorter than the wildtype genome).

[0152] Example 3

[0153] Whether the genomic stability of the HSV-1 vector depends on net genome size was studied. Genome stability of HSV-1 viral vectors varying in genome sizes due to deletions on non-essential viral genes were analyzed. Due to the requirement for maintaining the vector size within the limits for efficient DNA packaging, optimally to 100% of the wild-type genome length, these deletions were replaced by “stuffer” segments of non-coding DNA. To this end, a stuffer fragment based on HSV-1 non-coding sequences with similar GC content as wild-type virus was designed (see below stuffer sequence). In addition, the stuffer sequences were screened to ensure that they did not contain any open reading frames (ORFs).

[0154] Large portions of the HSV-1 genome had to be deleted to construct a reliable, nontoxic, and non-replicative DNA delivery platform for genomic medicine. However, the deletions resulted in vector genome size below the packaging limit. Sequencing experiments using Oxford Nanopore Technology (ONT) sequencing of vectors revealed that due to backbone engineering (Figure 3), when the size of the modified HSV-1 vector genome size decreased to 131.553 bp length (Figure 4a), the resulting vector underwent rapid rearrangements during serial passages in cell culture (Figure 5b). These manifested as genomic instability of the virus population, giving rise to high degree of genomic variability such as amplicon generation, nucleotide substitution, and / or duplicated genome regions after only a few passages. Therefore, these unwanted recombination events prevented generation of vector stocks with genomic homogeneity. These findings suggest that there is a relatively tight constraint on the amount of DNA, which can be packaged into virions, and that decreasing the genome size results in a sharply decreased rate of virus genomic stability.

[0155] To mitigate a risk of genome rearrangements and in a consequence vector instability, a HSV-1 pre-vector was engineered by inserting non-coding sequences of the “stuffer” module (Figure 4b), containing no open reading frames nor promoters’ regions. The modified HSV-1 vector with inserts of the stuffer (Figure 4c), resulting, after transfection in mammalian cells and auto-excision of the BAC module, in vector DNA closer to net genome size of the wild-type virus (153.533 bp) was genetically much more stable. Formation of any recombination by-products such as amplicons or duplicated regions of the viral genome in the modified HSV-1 vectors with stuffer (Figure 5a) was not observed. In contrast, “no stuffer” construct with genome size of 131.599 bp (Figure 3) generated vectors with an amplicon-like genome sequence of around 1.5 kb, containing oriS and the packaging signal (Figure 5b), repeated multiple times as can be seen in the overrepresented fragment in the sequencing data (>90%), which is likely a result of its concatemeric nature - generation of amplicons represents most likely a form of genomic instability due to suboptimal (short) vector size. Another undersized vector (size 138,012 bp) rearranged with duplicated regions of the genome (9,000 bp) to reach optimal size of (147,000bp) (Figure 5c). No such instability was observed with optimal size in modified HSV-1 vector shown in Figure 5a over repeated passages.

[0156] Example 4

[0157] To further investigate the size limits of the modified HSV-1 vectors to retain genomic stability, multiple modified HSV-1 vectors were constructed with various sizes, from different HSV-1 pre-vectors and stuffer size. These vectors were produced up to 10 times on Vero 7b cells and genomic stability was evaluated. Viral genomic DNA were extracted and sequenced using Oxford Nanopore Technology. Analyses were performed using the Geneious software. Genomes containing DNA region duplication or recombination byproducts amplicons were classified as unstable (Fig. 6, circle dots). Genomes without recombination events were identified as stable sequences (Fig 6, triangle dots). Recombination by-products amplicons and genome duplication correlate with shorter size of the genome (<142kb) (Fig 6, circle dots), while stable sequences without recombination events are associated with larger size of genome (145kb-154kb) (Fig 6, triangle dots). To conserve a genomic size of modified HSV-1 vectors between 147-154kbp regardless of the size of transgene(s), a library of BAC-HSV1 backbones with decreasing size of staffer were generated based on the 22kbp staffer fragment. The 6kbp, 12kbp, 15kbp or

[0158] 19kbp staffer seqaences below are fragments of decreasing sizes of SEQ.l, with a percentage of GC similar to WT HSV-1.

[0159] Non-limiting examples of seqaences snitable in the invention inchide the following.

[0160] SEQ.l - Staffer fall seqaence (22000 bp):

[0161] GC content: 68% ggtcgggcttggatttaatactaacgcgtaaatagtgagctttcacgcgcctccgcctgggagcgcagtctcggtggggatggcctctg tccggcgccgtcgccaggccggtcacgacgccctttttagaatgcaaggaccgtttactgagcc ttcgctcccgtctccacatagtgataaaggcacacagcagacgaccgcgtaaggccgtggacgtagcgtttggccaagacgtccCct gggaccgcgccggagacggcggcggaCggccctgcctccgGtaccgtatctccgcggacagtcggagtcgtagagttggctgac tactacctcgccccagtcgacgaaccggtacccctcgttcccgcaggccagacgcgcgctacgtcgccctactctccctccataacac caataactctcagtaacaggcgcgtccggcttctgtgcccgatctctcgccgcccggggcccctctccagtgtcccgacattatccacg gcccccgctcgtttgccccgctgctcggtcgttatgtcggcccccacatcgggggtgatcattgccgcgcccgggggggaaaagcg agcagccggcgcccagatcgaaatcgtttttgccctcgcagacctttgggtctcgcacctgaggggtgccccaaggtcgatcgccctc ctctgaaaatgaaccagaacagcagcggggagtaacccacgtggccccgaccaccacaatcgccaggctgcggaagtcgggccc acgagccgccctccacggcccccgcccgccccctctcggcgatgtgggttgctcgtggacgcgtgcctggaccaaccccaactcca gccccagtgtccccttttcgcggcaaagactggttgtccgtggccgaggacgtttcgttgcggcgaggtggacgttacgaacacgtcg atatatattaaaggtgcaggatgtgttttcgtgggcctcttccagtataagggttggtcgcctgcggtcctgacggccaagcgtccaacc gtttattcgccccagattatcttcccgctacgaccgcgcggcggagcaccgcctggttatacccgtgacgcccccgagttggtaccgta tgcgtgcgaattggtcggggccaaagcgggggagcggttttgaggtccagcgaattgagcgtgaagtttaccccggtatggggcgg aagacggcatccccgccgggttttctggtggtcatgtgagctcccagcgccgcccgggggtccgcacggggccgccgtttccaccg ccgttatacacgtagcccccagcggcaggagccccaccgcagaatctggttcacctacaatgccgctagggagagggtcaggctct ggcggaccgtggcgcgacggacaccgcgtgtgcgggctgggggtggcgggttggggctggggccgcggggctttgcgcgggcc cacgtcacggccaacctgcagatagatggcggtcacctgccagtctggaggtcggtgtggtcaccacgagtacctaggcgagtctctt tccgccgaccctccgtttgaggcgcgtgtactccgcgacgttacgggacgcacgccaccacggtagccgcgctgcgcggtgtgcgt ccggccgcttttatgccgaaggttagcgtggcgcgccagtcgcaacgccagcccccgtgtcggtccagcggcaaccctgggggac cctgatagttaaggacgtctctgttcggcgcggcgccggacagggcgaccacggactcgggaacgtggacatgggcaccggcgtg tccgagcccaaatgcaatgccgcgcctaccaccccccggtatacgacgaacccttggttccgacgttcctgatgcacgcgcgccaaa cgccagcgaccccacccccgacttcctcgtacggacaccaggtgcggaggacttgagttacccctcgggtctctaaagggggcgac tgcgccggccccgggtggtcgccgcacgtcctcgcgggagttccagtacgttcagggtgggggtgggcccccaccttcgttcaccc gcccgggacgaggggggtgctgcacgccacctactgggggaccaggcggcagcggagggcgggcgaaagccgtccgccaga gcgtgagcatggcggcgcctgtccccctttagccgcccacgcggcccgcagtagcgtggattacgtgcttccgcccagccgcattcc gctggcgctgtatgtgggtacagccgccgtcggttgcgcccctcgacgcggtggccgggcacaggctcttccgggagcggccacg aagccgcgcgcctataaaactgcggcccgcctacgcggagagcgtgcgcacctgcgggggcgcccggacccgggaggcctaat agtgacttggctccaaacacctgtccgccgtccggcgacgcttcgggtggggtccggctggtctgcgcggccccgaaccaagcggc gtcggggccgcgcgggtggccggaagcacctgaagacggcgcgacgccgagggttctgttgcacgagggggctgtcgggcgtc agcgcacaggtagggggggacgcgtgtgcgcccagcgccgaagcgaaatcaacccactgggacgcggggacggtggcgccgg tgtttctggggagacgtggacgtctcgctggccgttctgtcggagaccggggctcccggcggcggtggtctccgcaagagcgacag cgcgcaaatagcgcggtatcgtcgtgtccccgattccgacggggagcgaacgtctcggctaggttcggcccgagtgcgcacgcccg gtccggccatctacctcgcgtgggtgacgtggacccttcgggatgtaaagacttgtacttttgactacccgtactacttgggggtcgtga gcgccgggcgtggtggcaggtaaaacagcatctacgacggccagcaggctgggatcgaaggcgggggtccggggcaaacagcg gcgtggccgccacctggacggcgagcaagacccccccgtttccgcgggcgcctcacgggtaaaacccggtcggcaaacagctcg ggatccccccggacactgcgcgctcgcacgcaaccgcgtcgagcgccggggcggggggcggggtcaaacatgagcacgttgac gagtttgcttccgacgcccccggggttgtcggtgtgcgcaccgactcgtaggccacgacggcctggacgtaactaaacagaaccag ggtccggcgtccgcatcatctacgggacgtctcgatgtcggagctgcagcaggctggaagaccagcaagcaggtacttccagtccc gccatctcctccgggcaataaaaaacggcggtcgtgtccgccacgccccgcccgtcatctggttgtcaaacacggaaggcccgatcg gcacggcggttctgggggcctccggccgaaagccggtgaccgcgataagcgcggactaaagatgcgccgctcggggggactggt ttccctgctaaagtccacggccgtggccggcctgggcgcgcagagcgcgtactgcattttcacgccccggtccgtgctgtccgccgc gcgggttctggacctgcagggcgcgaactgcacacgctgttttctgggtccgtccttgccggcgccgcgctccggaagtcccgggg gggacggcctcctctaagggagggggcgcagtgtatacggcccccctgcacgataccgaccgggtcggcggcgcttgcttattgcc gttgacaaacgggggggtcaggcgaccttggctcctcaacgccactggcctgcgaggttccacgggggggagcgagctccgacag cgctgtgttccgggcacgcgtcctccaggcggaccgtgggtgttggtccgcgccgcttgctccgatttcgcgaccgcggacggcgg ggggccttgacgtcaccatccagtgtcgtccaccaacctctccatcctgacgatcaggcgctccacagggccgtgcctcaccgcgtg gtcgccaggacggcacgtcgctccaagacgtccaaagccgcgccgccccgacgcggcggagcaggtgcctccggataacccaat catctcccgggccgtcggagtcgtacgtgccccagtcgggacgcggtaccccgggcaccgcaggccagcttgggctacgtctcgc gagtctccctccagcctcaactctcagtttatcgcgcggatcagtacctctgtgcgtccagctcgccgcgacggccccctctcgcccac ggccgacattaaccaccccccgctccttggggcccgctgcgcacgagttatgtcgcgcgggccatcggggtcccccgttgccgcgtt ggtccgggaaaatagggcgggccggcgcgctgcgggaaatcgtctagcgcctcgcagaccgtcacctcgcaccgcgcgccccca aggagggcgccccctcctcacaaactgaaccagaccctattggggagtgttcctcggtggcccccccttacacaatcgcgaccaccc ggaagtccagggcgggcccccgccgccgccctctcatcggagtcgtgggttgctcgtcttgcctggactccccgcaactccagacca cctgtcccctcaccccagcaaagaccttccggccgtggccccacggttttcgttgcttccaggtggacgtcggagacacgtcgatgcg tgtgaaaggtgccttgtgtgttttcgtggactcgttccagtaactcggctcgcctgctttgcgcacggccaacgaggacccgtttattaaa cgcgattatcttgaacagacgaccgcgccctggagcaccgccttttttacccgtgacccaccaagttggtacgggcggcgtgcgaac gtgagggggccaaagtcatagagcggtttctgttgtcagcgaatccaggcggaagtttacccccccggcggaagacgcgagtcccc gccccacgccctggtggtcggcccggctcccagcctcccccgggggtctgcgagcgccgccgacgatagccgccgttaacaacat agcccccaccgaagggagccccacgttctcatctggtttgccatgaccgcgccccacgtcaggcagttgtcgccgtggcgcgacgg gcgcgtgtgtgcacggggggtggccggaacgggctggggttggggcgctttgcgggggggccgtcacggccaactatagggccc cgtgttgccagtcaaacgaccggtgtggcagcggagagtacctgcgtcactctctttcccgccgcctccgttgtattctgcgtgtactgc ggtctgttacgggccgggagccaccacggtgtcggcgctgcgcaccagcggtccggcccctccggtgccgaaggttagcggccag tcgttctaggagcccccgcaccggaccagcggcaaaacctggggaccccgccccgcgtctctgggtccggcggcgccgggcgcg ccgaccacgctagagagaacgtggagccactcaccggcgtgtccggacaactacggcaccacccacaagtgtacgacgacgcag ccgttccgacgcctcaagcacgcccccgcgcgccagcctgtttgtcccccgaccccagcctacgtgggccgcccaggtgcgcaag cgctgagttaccccgcgtgtccattgcgtcctcgggcgacttcaaatgccccgggttttttgagcacgtccccattcggcacgtcccctc gagggtggggggcaggccccacctgggggcccccgcccgaacatcagggggtgcggttaacacctactttctggccGggggacc aggcggcggcgggcgcgtcgcctctcttctaccactgtccgccagcatggcggcatggcgggcccgttcccccttccccgcgcacg cggcggagggctagcgtgggcattttgcttccgctgtaccgcattccgcgtttggtgtatgtggttgtacgcgccgtcgctcccgcccct cgaccgactgcccgggcacccggaggtccgggaggcccgacgaagatgctagcggcctataaaaaaggacgccgcctacggga accgcgtgcgcatgtgcaggggcgccccaccgcgggaggcctcctttgccaaacacaaccggtgccgtcgtcgaccgcttcgggg cctcttcggcatgtggtctgccgaaccaagccttgaggggccggattggggggccggaagcgtaggctgaagacggggtgtcgcc gagggcggtcctgcacgagccagtactcgggcgtctgcgggcaggtcgcacgacgacgcgtggactgggagcgccgacacgtcg atgaaatctccgatgtgggacgcccgcagagtggcgccgcaaatcacgccccccgtggaccgtctcgcctggccgtctgtcgggcg gattggctccctgggagacgtggtctccggttccgcgacagcgagcgcgtagcgcggttccggtgtgtccccagaagcgacgggga gccgatttctcggctagtatatgcccgagtggaccacgcggtccgcgctcggatgttgttccagtagccgtggaccgtctttcatgtaaa gcagctgggtactttttatgtctgtactaccccggggatcgtgagcaggttccgtggtggcctgaaacacagcatcttggccagccagc agatccgcgcgaaggccacatacgcggggcaaggaggggcgtggccggggtggtgacggcgagaagaacccccccgttaccttt gcgcctccgggctgcaaacccggccggatgacagctcgtcgaccacccggacaactttcactcgcacgtacgcgcgtcgagcgtg gcgtcggggggcggagcctgacatgagggccggtcacgagttttccccgcacgccccccgtggactcggtgtgggcgtgtactcgt agtagaccccggcctggacggagaaacagaatcacgcggccggcgtcccatgcctctacggggacacgggatgtcggacgacac gcaggctggagacggaccagcaacgcgctgcttccagtactgcgttctcctccagagcctaaaaaacgtttgtattgtccgccgaagc gggcccgtcacgggggggtcaaacagcaggtccccgatcgccgttacggttctggccgcctccggccgaaaacgctggaccgcga ggatggtggactaaaaacagggcgctcgggaacgccctttccctgctacgacacggccgtgcccgccctgggcgcggcgccccgt actgcagtggcggccccgcccacctatctgtccgcctcggaggttctggagccacccggcgcgaagctgctccgctgtttacttgccg tcctttggatccccgcgctcgacggacccgggggttgtagccctcctctaccaggagggcgcaggaccccccggcgcttgggaagg ggccccccggtaaaataccgacccccccggcgttggggttttgtgggggtgtcgcctgcgaccttgtccaggctgccaccacttaccg ggtgcgccaggtcggcggggagcacaaggggagcgccgcctccaggggtttccctgcttttgaacgttacgccctcgtctcggccg agttgctcctccacgcgaatgcgacccccaagcgcgcctgcagcagggtgctcgccacggcgatccgggaaggccagtaagggc cggggcttcgcaggcagcgatgtttctcccaaaaacacggcaactatgtacgaaggctcgaaaacacgctgcccaggcaaacacgc tgcatctgcgtgttgtggtcgtcctacagactcccgattaacgttcggctagcttggccgtgagatatacgccgcggcctcgtgcgccac aacagaagccgaccgcggtgagcatccggcttgtggagcagcagacggcgcgcatgacgcctccaggtgcgcctggataacaac cacccgccagggcgctacgacataggcgcggggagagggctccccaggggaccacgtgtactgcctgcgctccctcagcagccg ataagcgaccttctggactcgccgtacgcgaagccggggtcagccgccagcgtgccctcgcgggccatcccccccagtcgttgtcg cccagcgccttcggcagcgcctccggaggtggtgagccgatccctcgcgccctcggtcaccccaactctactgggggccgctggg ggccccgacgcggacgaggactggcctcaacgccacctgccaaatacagcggcccactagggtgcccaggcgggcggccgaga gcacggtgccgctgggtagaatccgatacgacctcgggcagcccccggagacccccaggtcgtccctcgcatccccctggggggc ttctggaccagctgaccgtctggtatcaaactccggggcctacggggagctaatcgcgatttcagacagcaccagaagtgccgctccc gtcttgcacccccgacgaacgttggccgtcgcgggcggcccgagcggccgtgcacagcctggcccaagataaggcggtccaccg cgtggtccggacgcgggccgccgtcccgaggcgtgggccggtgtgaaagttggcggcgcgactgcggtttgcacaggagcgggg caggcgctgtgagatccgcccctcgaccaccacgtccgtcgcttgccagtttcgtgccgggtatgccgaactgacgccacttgacgcc cagccagcgcggtccggcccgatatgaggagcgaccgaaaaggtcacgaccgcgctccggtctgatgctgcgggaccgtgaagta gcgccggcgctgtaatacagcgtacatcgccagctcggagatctctgcgagcgccacggtctggtccgagctgctcgcccgcggttt ccgggccaggggggttggggttggggtcctcgggcgcagccgcgggacgcgggtgcccgtggagcaagatcagccgggacga gaagagggccaggaacttgctgcccgcgaggccggaggacccagagaccgggccgctgccgctcgtctcctcttctcgcggggg cacctccaagcccagcagccgcgaggttaaggtgctccccagcgggtggcgggactccggagggcccaccagcaggcgtgcga cgccgccgcggcgttcagttgtttgaggcggggtggacggacgtcctcggcgggagagcggctcgctgggcgccccgagagggc agcagcagcagcgaacaccgaagcgcgtgcgctcggtgcgccaggccgaggcgaggggggcgagattggggccacgaccccg ctgttctcggaccgccggcacgtactggggaggccataattacgacgaggaggtcggtattaaggcgccgggcgtccagaggttcct gccaacaccccaagttttcgcacccgcctccccgaagagcgctggcgctggccttcgcgttcgctgccttcccgcctggctccagcca cgagctccagcttggccgccacgggcagccgaagactgtacacgcctgggatctgcgcgtgctcctcggggagccgccggggtcc cagccccacgccacggagcctcccgggagcgcgatccacgcggcaggccactaccacgcgcctagtccggctcgtagcgtccag cgcggagctgctccaggcggactcgacccggcggtcgacggctgcggcggggtactgcagcctggcgacgtccgcggccgcttg gcgaacaccccgtgcctggtcatccagatcaccacgcccagagtcagtggggcgcccccaccgccgccctaatctggggggggtt ggcggccgcagcagcacgagcacctgtacgtacggtggctgcgatccaacatacaattgacccccgggtcactcccacgtcttctcc gtgtacgacggccggctgcgtcgctcaactccatccggacactaacccccccaaggccacgtcgccgagtacagatcctcgtacctc gtctagccggccccgcggggccgcggcgctgctgttcctcctttggggggcagccgaggggttttcctgggggcgccggtccagta ccgatctggaggccgcagcggtctcccggccaccaaactggcactttatacacagatgtcagctacgcggggtgggcgctcctgatg tgcggggttgtcgtggatccccgggggtgtagggggtggccggtgccggtgatgttaacggtgcgcacgcgtatgcaatttacgacg aggtgggagggcagcgcgttaaaggggaaaccatcccgggtggcggcggggccggacctccgcacctggtgatgcactggccg cgtttgaccgcgggccggggtcccgcggggttcattccacagggctcgaggcgggccggtggtcggcgccgtcacgtcgtgcgcc tcccagcagcgccgggggcgtctttacgtgccacgcgcgctcgaacgggcgcggggccgccggggtgagtcccagtcgtcacag gaccctctttttaatatctatcgccgagcgggaggagatgccccagggctaagaggtccattgggtacgggaccaaagcgccggcgg cgcaccccgcgcggagatcgactagcggcggcgcctctgcggtcgccaggggactccagaatcgcgcctcacgacacccacccc tatgacctgttggcgcgaatctccacattggagacgtccgcggagcgggcacagctcgcccccactcccccgtatacggactccatct ttcggcatcgcgcaaggggcaggctggaagactgtgcgttcgcggggcgtgaggatggcgcgcctctagctcgctgttcgcatctttc ctgtgcggcgcaccccgaccgcggcggcgcctctccgcggcggcggcggctcccaggcctccgacccggtcctcacgcctggca gcacgcggaggtgtccttgaacggagtactactgcaccgcgttgtcgccgccgacgccggtgtgactgacggcctcctcgcggcgt cgtcgtcggggcgttccatccgggtatctcgcttcccccggcggagtcgtcgtccaggcctccgcgtcatgccgcgcgaaatcgccg gcacgccatggacccctcttcttccccgcccgacggcccggtgcgtgcccgccagcgccggcacgccatgacccctcttcttccccc gcccgacggcccgcgtgcgtgcccgccaccagcagagccagctctggagtttgtgcaatccaggtcgggcgcagtactccttaacc ggggcgcgaactggtttgtgtgtgcggggtgtcgcctgcggcgaccaggctaaggccctaccgggacctcgaggggtcggcgacc gccaaaggggccgcgggtactccaggatgatgtctgcttttacgccacgccctcctccgcgctgttgacaaacagggccgaattttaa cacagaagcgccaccagacggtgaccgtatccacggccagtacgagcttcttcggggtgaatgttggcggcgatgtcgcggacatc ccccaaagcgagctccgaccacgcttggggcgccaaacacgttatacagcgtgttcgaattcgcaggcccagcgcacagtaacgtg gaccaccgttggcctcgtcaaagcgccgggcgggtacgccacctccagggcgaccgggccaacccaatccggtctatgagcagca gcagctcgtcggctgacgcggccgttgcgcctttcagtttacaaccacccgcacctacgaccggtgcgtttagcgtgcccggtgcgcg gggagactgtttccagggggtcgggacgccagcgagtcaagctcagcatccttaacgatagacgtacttaactcgcttctggttaaag ccgcgtctgggcagcccggaggtcacctcgcgggcctcgtgcagtcgtacaggggaagcgcctcgtgctcgcgccatcgccacgt cgatccaaggagttctcgcgcccgccctcccccaatcccccccgagggccgagccggtttcgacgcacccccccaggactggaag accccctggcaggccgaaaacagcaggcgccactgccctggtcgaacaccgaggcccagaccgccgctgcggcccgtatccgat acagccttgcacccaacacccccaaaccttccccgctgaagcccccaggctttgccgtccccctgaccgaccctccggtatcaaactc ttttacggggcgcggccccgcaggcgtgcctgggcgatttccgacgaagcaccagaaagcacaaaccgcgccctccccgtctgac gatctgcacccccgacgagggttggccgtcgcgtggcccgagcggcccgtgcacacctggccgtggataaggcggtcgcagtggt ccgtctcgctccgccgtctggctgccgtgggcctctgaccagttggctgtggttaactgcggtttaaggacggggtcagggggtgctgt gagattcgcgctcgaccaccaccaagcttggccctcgaacttcgtgccgggccaccgaactgacaaaaaattgacgcccacaaccc ggtccggaggtcgtgaggagccagaacgctccggtcacgttccgggaccgctggagcagcgccggtgtcccataatacagcgcgg ttcgccagctcgctcagcgagcgctggccgtctggtccagacaggcggcagcgcgcagctcgcccgcaagcgccaggggggacg ggggttggggttggggctgcgccattaccacgcgggtgggggcgggagcaatacaccgccgggacgaggccgtggccaggaac tgggggcccgcgaggagggcaggacccagagcgaccgctgccgacccagtcctcttctccccttgcacctcccccttagcagccgc tgcaggtgctcgcgaattgggggtggcggaggcgcgagggccgtcgggcaggcgtggtacgtgcgccgcggcgcagtattgaga cggacccggggacggacggccccgtcggcgggagagtactgggcggcttggcggatgggcaggtagcgcgtgcgcacgcggta gccaggcccagaggcgggggctgggttggtggccacgggatttactgttctcgttcctccggcacgtacctgcaggccaagccgcc aggatgacaatgacttaaggcgtcgacgccagagggtagagccggtgtagtggcagtacggtgccgcgcgggtgttaacggtcagc gagtatgcagtttaggggaggtggcggacgacgcagcgcgttggggaggaaaccatctccttgcggcgggtgacgtcctccgcaca cattcgatgcactggaccacgtgaccgcggagcggcgtcccgcggaaactccattccacagtacctgcgggccgcgcgggtcggc gccgtggccgcgcctccagcagggggaaaacccactgcctgctgcgcctgctcgaacgcccccgccgccggagtcgtcaggatgt cccagtgggtggaccctgttcgaccctatatagtttggtggaggaaccggcgatctagggcggagcgcccattgggtggggttcaaa gcgcttcgacctgacgcccgcccccgactacgcgaccctggcgccgcggggggcgacggggaacagcggaatcgcgcctcacct cccaacgcctcccctgttcacattgccccgccgccgcggagccgtagagctcgcccccgagcccactcccccgtatctctttgtgctgt gccgatcgcgcaaggcggcgctggaagactgccagtgcggggcccgtccctcgcggctcaactggctcgctgtcgtcatccttcctg tgtgtagttcccgaccacggcgcgcctctgcgtggggggccgcggagctcgcctccgattcgtccgcctcacgccccctacgcggat cgggcgatacctcctctcatacctccatctcgtcgccgcggatcagggagtgggcggaggcctcctcggcctccatcggcgtcggcg tggcgggaccaaaaagcttccccccgggagatcgtcgtcgtcaggtgcgtccggggtgtacacgcgcgaaatccctcgccagctcg ccgtccttcttccctcttctcccgcgcctcgcgtggcccgccagcgcctggagccagctgtcgcggtgcaagaacttctcgtgcgggg gggcttgcggcgacctcggcgcaggccctgggagcgcctcgaggcggactggcgccagccgcggtttggtatagatcgacggga gtcagcgttgggacgccacggcgagctccgcgctggtgcggtacagggctgggtgcaaacacaggtcgcgcgcaccagaccgtg gtcgtatccacgggagggacgattgttaagccagctttttgtacaacgggtgaatgagctggtgtcggccgacgtaaatcccccaaaa cacgagctccgacaacgcgggcgccctccgtcgggttatacaggtcgcccgaattcgccaatgaagcgcacagcggctgggacca ccgcgctggtcgtcaaagtcgccggggcgggtaacggtactccagggccgcgtcgccaacccagcgccactatgagccggtagta gctcgtcgggatggcgcggccgtctggtttttcagtttcatggcccgcacccccagggccggtgcgttgcacgtaactggttccgagc agactgtttccggcgtgtcgggaccccggagagtcaatggcgtgatccttaaccgctggctgtacttaatgtagcgctggttagggcga ccgtctggggcgttcggaggtcacccccacagcctcgtgggcacttcaggggattttccgccgtgctcggcccccagccacgtcctc gccgaggagttcgttgaggcgccctcctcgccggccccccgaccgggttgccggtttatatattcccccccaacgggggaagacccc cacggagggccgaaacggccatcgccacaccacgccagtcgaacagcatgttccagaccaccaggtgcggcccgtggccgagca gccttggagtctgccctacacgcacaaaccttcccacaccaccccccaactgaccccgtccccctcgtagtaccctccgggggtccac tctttgtggacggcggcccgacccagaccgacgaacgcggacgcacaggccatcaacacgatcttgattggcgacgaggtgcgcgt gcgtcgcgtgccgcctcggcccgtcaagcttggccgtgccccccctcggtcgcaggcgtcccgtctcgctaacggactggctgcgg cccgccctctgaccaggcctctgtggttagggcggtaaggagtgcgcgcccagggggtgcgtcacagattcgcgggccgcaccac caacagcggccctcgaacacgggcggggccacctggagctaaaaaatcagaggccacaacccgcgcacgaggtcggcgttggtc cagaacggcgtcggcactctggggcgcgctcacgttcggggatcgctggagcctggtggtgtcccacggtgcacgcgcggtgttgg gggtcgctcacgttgttggcatggccgcggccccagacagaagggcaggcgcagctggggcccgcaagcgggaaagaggggga cggttttggggttggggctggggtcccggtgccgaccaaccattacctctcggatgggggcgcgcctggtgacaccgcgcccctag gccgtgcacacgaactgggggtagctcaggagggcgtgtcgtcgagcgacccggggttgacccagcggctgatctccccttccctc ccccctgtgcgtgtaactgcaggacgagcccgaatgtggtttagctgaggcgcccgcgcagccgtcgggcgcgtcttggtacgtgcg ggccgcgcagtatgatcaggacccggtcaccttccggccccggcgccccggagtacacgataaacttggcgtgagcggcgtagcg cgtgaggcccaagaaggcgggggccgcggtagcgcacgtgaggcggagcagcgcgttggtctcggtgcagggatttaccgcgga gttcctcactgcctcgtacctgcggctcgtagccgccgaagggaccaatgacgagggcgagtcgacgcagacgcatagagccctga gtcacaggaaacagaaacgcgtcgcccctctccagctcttggaggattctcgactccacgcgcgttttcctccgcagttccaggcgact gcgtgctctccggtgacgttcatcgtcggcctggcactccggccgcaggcaggggcgggcggttggaccacgcggagcaagctgc gccagccgagttctcgtaggagagcgcggtgctgtaccggagtgcagtggtggcgttgtgaaacaggtaggccggtacacacccgt tgtcggcccgccttccgtggcttttttcgagctgacccagcgtccgtggcctagggcgaactcgatggaggcttgcgtggcgatgtgg cactgactcaggatatcctgtctgctctcccgccggccgcagcaggcatttggcacacccagtgcgttccggcgcgggacccgaggt cgccctcgatcgccctggcgtcctccagttccgcctttgcagagccaaggaagtggccctccgcgatcggtaacgcgaccaagcgg gggcgctggggtttggggtccgagggcgtgacgtcccagctgggggcggcgcgctcagtaacggccgtgggcgcgaagactcat cgcgcggaacgagctgacgccggtccgggtttccgctttatcctctcgggcatcgtttcccgccgcgaacccgacccgcgcctcgcc cgcccaggcggcgccggttcggaccaccagctcctaagcgccccgtcgatccaggaccaacgccgctaccgatgttccagaacaa acgctgctccgctaaaacctggggtcacgcgggcagtaaaacacgtccgcgggtcgttaacatcagcgaagcggcggtttagggtc agcagcgacgaccaaaccgactggggatcaccgacgctccttctcgtttccgacgtcctgcactgccattcgctcctatcacacgagc cgggccagcgcggcgggcgcggcgaaactcggccgtggtacctgtcggcggttgcgcggggtcgactcgtggcccgggccttga gcagggcgtcggccacccacacagcatgggcctggccgaccagacccgccttccagctggtcgtcagacgcgagaacttctcccg ggcggggtctgaccatacatcgaccgggacaccatttggtcccccctctccggcgattggagggaagcgcacggactcgggggttg atacgggacttcgaaactacccccgcccccgcgtattgcccttggtgtcggggggggcctgtggggagagcgggtggtccgtgcct cgcgctaccgctcctgttggtataggtcgtgcctggcgggcggccgccgcttgatcatgtagaaatcccgcaggagccccacgcaag gtgccccagctgcctgtgccgcggcctcacgggcgtccggcccgcgccagtgccgacgaacgtcccttcaaccactaactggccgc cgggcatccccgcggaacactgtagttgtttatgtggcgcgctgggcagggggggggcgcggggcggagctcggcaggcgccgt cttctggtaagcccttttattgatctggcgtctggagcggcatctcccggaaggctctccaaggcgaccaatcaggttggcaggcgggc ggaggacccggcctccagcgggtctgcgtggcgggcggcgaaagctgggggcgaagagatccgtgtcctgaggtggtcggcgtc ggtgtacagcagccgcccctcgggcgatttgccgtccgttccctgtcttctcccgccctccgcgtgcgtggtcggcgcctgcaggcga cgtcgcggcgagacggcaatagttattcggagtcgtcaccacgccgccgccctccttcggggtgccgctgtcgggacaggccacgt acgccagcgaggcgaccacccggctcgcgcgcgtcgacgtcgactccccgcctccgggagcctgtgggtccggcaggcgggtga actccacggccgagggaatggacgccttgctgggttgcggaggtggcgccggatgggacgctccacgactgctgcccgcaccgtc ccccgacgacaggggacggcctcctgaaccgcagacgagcttctcaactgggtcagccaggcgacgaggggtcgctggcgcagg tgagagcccgtccggactgaaggtagattgccaagtttggtctgtagcaaatgaatgacggttcttggtcgtaatccaggataaattcga ggccacacgcgtcagggacttttggagtcgccctgcgattttcgagcgccgcgtttgttgggaacttgggtgtgccgatacagccgtcg ggggccccaaacgggtctccccgcccccgccttgtttgaggcaggaccacgagcacaaacgtgcgagatcgagtggcgtctaacc cgactgcgcgctgcggtcgtccctctacgttgcggggtagcgtccgggcgcttggcagcgcccggcgggtccgctgtcctcgcggt ccgcctggcgtcgcgggacagggcctgcttccggccccagcgcagccacccaaagtgccccgaccccgagagtgggggggcct gggtccgcgtcctgcagggcgccgaactgacagccaccgggccgtgtcacgatcaccgcgggagcggtgcggcctgggttggcg tcgggtccttcgcgtttaagcttggtctcccatcagcagtttccaatcccggccccggggaatcagaagcggagcgggctggggacg gggacggcgtacgagaagggccagggcgaggtgcggcgcgctttagcgaccgacgcggcaccaacaacgacaccgagccggc ggagagccgccgccctcaccaacctgttcttacgtcgccggtaggggcagacgccccctgctccccggaccccggcgccggaacc gagccccgcgggggcgtggccaagccccccgcccctgggcacgcgcacggcgatcagagggtcagtggtgtttcctcatggccc cttatcgggggttcccaccaaacctgcggccaacacaacggtggatgcctctctcaagatgggggcccgacggcgtagtggcgccc aaaacgtcctggagaatcaatctcgccgccagctcactactcggaaagcgaagactattgggttaaccccacgatgcctcgacggaa acctcgttattcagtcccgcaaggacatgaagccgtgttggtcgcggaacacgcttaggacgacgacctttgccaaggttctgcccgg gccgtgagtgcgtccggacagccgcttagggcgtccgcacgccacgctgatcgccagacaacgccgctatttaacaaacatccggg gggacttgacccagctgctcaaaacctcctggcgggccctgcttcccgggaatggggaagcgcctggtcccgatttgtacgtgtacgt gtagtacagcacgttgaggtcgccgcgaaggcccgggacggaagaccggctgggtatggggtcgagttgtttgaacacccagacg ccggtgagcttgtagccatagccaggtcaagccgctcgtcagccgcgtgacccggcgcgctggagacggcggcggcctgggcgc ccggcgtggaggaaaggcgaagaaggccgccgccccatgatgaaccgggcgagctgggtgcggggccccaggggcagtactttt acatgaacaggcgcgatgctcgaatatcaagcaggctcacaaaccggcattaaccggttcctggacctggctgtccctcggcgacct ggggtctgaagcagcgacacgctaggtcgcgacggctcgtagacgctgcttctggcgctgcaccctgggtggcggctgttgcgtgta caacatcatccagcccagcagctgcgcgttcgggcgcacgctggttcagcgcattgctcgccgagacgtctatccccaatcgctccg gctcccagtttgtaacttcgacccgccgtcagatacgtggcttccaaggcgcgcgtggccgccgctgggaggggtcgtcaacggcg ccgtcgacgcgggggcgtccggactcaaccgccgaacccacggccccgtcgatcgtgggcagcgaacagatcctggcccctaca cgaaaattacccggtcggacccccgccggcgtggcggctgcggtgtgcgcggcgccagggccaaaactccggctccctcccagg gcaccgacggtcagccccgccccccgggcgcgccggcagcacggcccggaacgcagccccgggcccccagggcgtattcgag aaacggcgccaggacagggaggcggcccaaccgtcccgtagtcccagacagcttcagcgccgtcaaacggctcggcctatggctc ctcggtggtcctggtggtgcgccccctccctgtttgtcggggtggggagtgggtgggtgaatcgtcggtgccagaccgacaggagtc gaacggatacgcgaggaaggcccgcttcagcattgtggaccggggaccgtctgccgggatcatggggagctgccccggaagccg catccgcgatgacgcccgtggcgtagtgccctgaataccatcgccgcgggggggtcttggggactgggcaacggcgcgggccag ctccagacctggtttctggtgccctggacgatcgcgcagtagcgaggcctcggtcagggccgtggcctactggcctcggagggggt gggaagcacccgggtgattccaggagagggggagccccaacacattgaccgcgtgggcggccgcgctggcgaaaagtaggctg cccttgttatctgagtcagcggcccgccagttgggacggccgtatgtccaagaacggggccccggagtccaccttgatctcgtggcg ggtgtaaagccgttggcgaccgcggcaacacgaccaggggcgcgggttcatcctggacaacccccgtgtaaacacagctcctgtac gtcgcgcgtgcgtgcccctgttaacccaagggccctccctggggctggggctggggctcgacacctgggccgccagctcgagtgtc tgcgcgccatggccgtggcccgggacgacgcgttacatctacaacaactaccctttagtatatcgcgacagttggtcgtcgacgattgc agctttctgggggcgggcgcgctctggttcgaggacgaccacctgcccccccgggccgctggctgaagcgggcgagcagggccc agctgccggacgctggcgcaagacggggtgtagtacagactgcgtccctgacctcgttttgtgttatatcgccgtggtggccccggtg ctggcgggccacccgcgcggcagagctcgacccccccgagccgcgcgccagcccgggcggccgggggcggggccaggtccc gcgtggacaatcttagtaatctcgagatgttgacgacaacaacgcccttcaccgccagggcattatcgacgcacacaccctagcgtga ccccgaggattcggccctcgcccgatttacagcccccagtgtctgtccccggccgacgcgtgtcggtgggtttggacgccgtcaaga actgttatcccgcgggggcgcctgcgtcggccctgcgggccaggaccaggggcggccagggccgccaggagcacgccccgcaa aaacagacacctcaagcgcgtacgcagttgcttcagccactggggcgagcgtcaccgtatcccccagaccgcagtaccatcgtgttt gccgagctccggcccgggaatcgggccacaaagcggcccccgattattcgcc

[0162] SEQ.2: 19kb Staffer

[0163] GC Content: 68% ggtcgggcttggatttaatactaacgcgtaaatagtgagctttcacgcgcctccgcctgggagcgcagtctcggtggggatggcctctg tccggcgccgtcgccaggccggtcacgacgccctttttagaatgcaaggaccgtttactgagccttcgctcccgtctccacatagtgat aaaggcacacagcagacgaccgcgtaaggccgtggacgtagcgtttggccaagacgtcccctgggaccgcgccggagacggcg gcggacggccctgcctccggtaccgtatctccgcggacagtcggagtcgtagagttggctgactactacctcgccccagtcgacgaa ccggtacccctcgttcccgcaggccagacgcgcgctacgtcgccctactctccctccataacaccaataactctcagtaacaggcgcg tccggcttctgtgcccgatctctcgccgcccggggcccctctccagtgtcccgacattatccacggcccccgctcgtttgccccgctgc tcggtcgttatgtcggcccccacatcgggggtgatcattgccgcgcccgggggggaaaagcgagcagccggcgcccagatcgaaa tcgtttttgccctcgcagacctttgggtctcgcacctgaggggtgccccaaggtcgatcgccctcctctgaaaatgaaccagaacagca gcggggagtaacccacgtggccccgaccaccacaatcgccaggctgcggaagtcgggcccacgagccgccctccacggccccc gcccgccccctctcggcgatgtgggttgctcgtggacgcgtgcctggacc aacccc aactcc agcccc agtgtccccttttcgcggc a aagactggttgtccgtggccgaggacgtttcgttgcggcgaggtggacgttacgaacacgtcgatatatattaaaggtgcaggatgtgt tttcgtgggcctcttccagtataagggttggtcgcctgcggtcctgacggccaagcgtccaaccgtttattcgccccagattatcttcccg ctacgaccgcgcggcggagcaccgcctggttatacccgtgacgcccccgagttggtaccgtatgcgtgcgaattggtcggggccaa agcgggggagcggttttgaggtccagcgaattgagcgtgaagtttaccccggtatggggcggaagacggcatccccgccgggttttc tggtggtcatgtgagctcccagcgccgcccgggggtccgcacggggccgccgtttccaccgccgttatacacgtagcccccagcgg caggagccccaccgcagaatctggttcacctacaatgccgctagggagagggtcaggctctggcggaccgtggcgcgacggacac cgcgtgtgcgggctgggggtggcgggttggggctggggccgcggggctttgcgcgggcccacgtcacggccaacctgcagatag atggcggtcacctgccagtctggaggtcggtgtggtcaccacgagtacctaggcgagtctctttccgccgaccctccgtttgaggcgc gtgtactccgcgacgttacgggacgcacgccaccacggtagccgcgctgcgcggtgtgcgtccggccgcttttatgccgaaggttag cgtggcgcgccagtcgcaacgccagcccccgtgtcggtccagcggcaaccctgggggaccctgatagttaaggacgtctctgttcg gcgcggcgccggacagggcgaccacggactcgggaacgtggacatgggcaccggcgtgtccgagcccaaatgcaatgccgcgc ctaccaccccccggtatacgacgaacccttggttccgacgttcctgatgcacgcgcgccaaacgccagcgaccccacccccgacttc ctcgtacggacaccaggtgcggaggacttgagttacccctcgggtctctaaagggggcgactgcgccggccccgggtggtcgccg cacgtcctcgcgggagttccagtacgttcagggtgggggtgggcccccaccttcgttcacccgcccgggacgaggggggtgctgca cgccacctactgggggaccaggcggcagcggagggcgggcgaaagccgtccgccagagcgtgagcatggcggcgcctgtcccc ctttagccgcccacgcggcccgcagtagcgtggattacgtgcttccgcccagccgcattccgctggcgctgtatgtgggtacagccgc cgtcggttgcgcccctcgacgcggtggccgggcacaggctcttccgggagcggccacgaagccgcgcgcctataaaactgcggcc cgcctacgcggagagcgtgcgcacctgcgggggcgcccggacccgggaggcctaatagtgacttggctccaaacacctgtccgcc gtccggcgacgcttcgggtggggtccggctggtctgcgcggccccgaaccaagcggcgtcggggccgcgcgggtggccggaag cacctgaagacggcgcgacgccgagggttctgttgcacgagggggctgtcgggcgtcagcgcacaggtagggggggacgcgtgt gcgcccagcgccgaagcgaaatcaacccactgggacgcggggacggtggcgccggtgtttctggggagacgtggacgtctcgct ggccgttctgtcggagaccggggctcccggcggcggtggtctccgcaagagcgacagcgcgcaaatagcgcggtatcgtcgtgtc cccgattccgacggggagcgaacgtctcggctaggttcggcccgagtgcgcacgcccggtccggccatctacctcgcgtgggtgac gtggacccttcgggatgtaaagacttgtacttttgactacccgtactacttgggggtcgtgagcgccgggcgtggtggcaggtaaaaca gcatctacgacggccagcaggctgggatcgaaggcgggggtccggggcaaacagcggcgtggccgccacctggacggcgagc aagacccccccgtttccgcgggcgcctcacgggtaaaacccggtcggcaaacagctcgggatccccccggacactgcgcgctcgc acgcaaccgcgtcgagcgccggggcggggggcggggtcaaacatgagcacgttgacgagtttgcttccgacgcccccggggttgt cggtgtgcgcaccgactcgtaggccacgacggcctggacgtaactaaacagaaccagggtccggcgtccgcatcatctacgggac gtctcgatgtcggagctgcagcaggctggaagaccagcaagcaggtacttccagtcccgccatctcctccgggcaataaaaaacgg cggtcgtgtccgccacgccccgcccgtcatctggttgtcaaacacggaaggcccgatcggcacggcggttctgggggcctccggcc gaaagccggtgaccgcgataagcgcggactaaagatgcgccgctcggggggactggtttccctgctaaagtccacggccgtggcc ggcctgggcgcgcagagcgcgtactgcattttcacgccccggtccgtgctgtccgccgcgcgggttctggacctgcagggcgcgaa ctgcacacgctgttttctgggtccgtccttgccggcgccgcgctccggaagtcccgggggggacggcctcctctaagggagggggc gcagtgtatacggcccccctgcacgataccgaccgggtcggcggcgcttgcttattgccgttgacaaacgggggggtcaggcgacc ttggctcctcaacgccactggcctgcgaggttccacgggggggagcgagctccgacagcgctgtgttccgggcacgcgtcctccag gcggaccgtgggtgttggtccgcgccgcttgctccgatttcgcgaccgcggacggcggggggccttgacgtcaccatccagtgtcgt ccaccaacctctccatcctgacgatcaggcgctccacagggccgtgcctcaccgcgtggtcgccaggacggcacgtcgctccaaga cgtccaaagccgcgccgccccgacgcggcggagcaggtgcctccggataacccaatcatctcccgggccgtcggagtcgtacgtg ccccagtcgggacgcggtaccccgggcaccgcaggccagcttgggctacgtctcgcgagtctccctccagcctcaactctcagtttat cgcgcggatcagtacctctgtgcgtccagctcgccgcgacggccccctctcgcccacggccgacattaaccaccccccgctccttgg ggcccgctgcgcacgagttatgtcgcgcgggccatcggggtcccccgttgccgcgttggtccgggaaaatagggcgggccggcgc gctgcgggaaatcgtctagcgcctcgcagaccgtcacctcgcaccgcgcgcccccaaggagggcgccccctcctcacaaactgaa ccagaccctattggggagtgttcctcggtggcccccccttacacaatcgcgaccacccggaagtccagggcgggcccccgccgccg ccctctcatcggagtcgtgggttgctcgtcttgcctggactccccgcaactccagaccacctgtcccctcaccccagcaaagaccttcc ggccgtggccccacggttttcgttgcttccaggtggacgtcggagacacgtcgatgcgtgtgaaaggtgccttgtgtgttttcgtggact cgttccagtaactcggctcgcctgctttgcgcacggccaacgaggacccgtttattaaacgcgattatcttgaacagacgaccgcgccc tggagcaccgccttttttacccgtgacccaccaagttggtacgggcggcgtgcgaacgtgagggggccaaagtcatagagcggtttct gttgtcagcgaatccaggcggaagtttacccccccggcggaagacgcgagtccccgccccacgccctggtggtcggcccggctcc cagcctcccccgggggtctgcgagcgccgccgacgatagccgccgttaacaacatagcccccaccgaagggagccccacgttctc atctggtttgccatgaccgcgccccacgtcaggcagttgtcgccgtggcgcgacgggcgcgtgtgtgcacggggggtggccggaa cgggctggggttggggcgctttgcgggggggccgtcacggccaactatagggccccgtgttgccagtcaaacgaccggtgtggca gcggagagtacctgcgtcactctctttcccgccgcctccgttgtattctgcgtgtactgcggtctgttacgggccgggagccaccacgg tgtcggcgctgcgcaccagcggtccggcccctccggtgccgaaggttagcggccagtcgttctaggagcccccgcaccggaccag cggcaaaacctggggaccccgccccgcgtctctgggtccggcggcgccgggcgcgccgaccacgctagagagaacgtggagcc actcaccggcgtgtccggacaactacggcaccacccacaagtgtacgacgacgcagccgttccgacgcctcaagcacgcccccgc gcgccagcctgtttgtcccccgaccccagcctacgtgggccgcccaggtgcgcaagcgctgagttaccccgcgtgtccattgcgtcc tcgggcgacttcaaatgccccgggttttttgagcacgtccccattcggcacgtcccctcgagggtggggggcaggccccacctgggg gcccccgcccgaacatcagggggtgcggttaacacctactttctggccgggggaccaggcggcggcgggcgcgtcgcctctcttct accactgtccgccagcatggcggcatggcgggcccgttcccccttccccgcgcacgcggcggagggctagcgtgggcattttgcttc cgctgtaccgcattccgcgtttggtgtatgtggttgtacgcgccgtcgctcccgcccctcgaccgactgcccgggcacccggaggtcc gggaggcccgacgaagatgctagcggcctataaaaaaggacgccgcctacgggaaccgcgtgcgcatgtgcaggggcgcccca ccgcgggaggcctcctttgccaaacacaaccggtgccgtcgtcgaccgcttcggggcctcttcggcatgtggtctgccgaaccaagc cttgaggggccggattggggggccggaagcgtaggctgaagacggggtgtcgccgagggcggtcctgcacgagccagtactcgg gcgtctgcgggcaggtcgcacgacgacgcgtggactgggagcgccgacacgtcgatgaaatctccgatgtgggacgcccgcaga gtggcgccgcaaatcacgccccccgtggaccgtctcgcctggccgtctgtcgggcggattggctccctgggagacgtggtctccggt tccgcgacagcgagcgcgtagcgcggttccggtgtgtccccagaagcgacggggagccgatttctcggctagtatatgcccgagtg gaccacgcggtccgcgctcggatgttgttccagtagccgtggaccgtctttcatgtaaagcagctgggtactttttatgtctgtactaccc cggggatcgtgagcaggttccgtggtggcctgaaacacagcatcttggccagccagcagatccgcgcgaaggccacatacgcggg gcaaggaggggcgtggccggggtggtgacggcgagaagaacccccccgttacctttgcgcctccgggctgcaaacccggccgga tgacagctcgtcgaccacccggacaactttcactcgcacgtacgcgcgtcgagcgtggcgtcggggggcggagcctgacatgagg gccggtcacgagttttccccgcacgccccccgtggactcggtgtgggcgtgtactcgtagtagaccccggcctggacggagaaaca gaatcacgcggccggcgtcccatgcctctacggggacacgggatgtcggacgacacgcaggctggagacggaccagcaacgcg ctgcttccagtactgcgttctcctccagagcctaaaaaacgtttgtattgtccgccgaagcgggcccgtcacgggggggtcaaacagc aggtccccgatcgccgttacggttctggccgcctccggccgaaaacgctggaccgcgaggatggtggactaaaaacagggcgctc gggaacgccctttccctgctacgacacggccgtgcccgccctgggcgcggcgccccgtactgcagtggcggccccgcccacctat ctgtccgcctcggaggttctggagccacccggcgcgaagctgctccgctgtttacttgccgtcctttggatccccgcgctcgacggac ccgggggttgtagccctcctctaccaggagggcgcaggaccccccggcgcttgggaaggggccccccggtaaaataccgacccc cccggcgttggggttttgtgggggtgtcgcctgcgaccttgtccaggctgccaccacttaccgggtgcgccaggtcggcggggagca caaggggagcgccgcctccaggggtttccctgcttttgaacgttacgccctcgtctcggccgagttgctcctccacgcgaatgcgacc cccaagcgcgcctgcagcagggtgctcgccacggcgatccgggaaggccagtaagggccggggcttcgcaggcagcgatgtttct cccaaaaacacggcaactatgtacgaaggctcgaaaacacgctgcccaggcaaacacgctgcatctgcgtgttgtggtcgtcctaca gactcccgattaacgttcggctagcttggccgtgagatatacgccgcggcctcgtgcgccacaacagaagccgaccgcggtgagcat ccggcttgtggagcagcagacggcgcgcatgacgcctccaggtgcgcctggataacaaccacccgccagggcgctacgacatag gcgcggggagagggctccccaggggaccacgtgtactgcctgcgctccctcagcagccgataagcgaccttctggactcgccgtac gcgaagccggggtcagccgccagcgtgccctcgcgggccatcccccccagtcgttgtcgcccagcgccttcggcagcgcctccgg aggtggtgagccgatccctcgcgccctcggtcaccccaactctactgggggccgctgggggccccgacgcggacgaggactggc ctcaacgccacctgccaaatacagcggcccactagggtgcccaggcgggcggccgagagcacggtgccgctgggtagaatccga tacgacctcgggcagcccccggagacccccaggtcgtccctcgcatccccctggggggcttctggaccagctgaccgtctggtatca aactccggggcctacggggagctaatcgcgatttcagacagcaccagaagtgccgctcccgtcttgcacccccgacgaacgttggc cgtcgcgggcggcccgagcggccgtgcacagcctggcccaagataaggcggtccaccgcgtggtccggacgcgggccgccgtc ccgaggcgtgggccggtgtgaaagttggcggcgcgactgcggtttgcacaggagcggggcaggcgctgtgagatccgcccctcg accaccacgtccgtcgcttgccagtttcgtgccgggtatgccgaactgacgccacttgacgcccagccagcgcggtccggcccgata tgaggagcgaccgaaaaggtcacgaccgcgctccggtctgatgctgcgggaccgtgaagtagcgccggcgctgtaatacagcgta catcgccagctcggagatctctgcgagcgccacggtctggtccgagctgctcgcccgcggtttccgggccaggggggttggggttg gggtcctcgggcgcagccgcgggacgcgggtgcccgtggagcaagatcagccgggacgagaagagggccaggaacttgctgcc cgcgaggccggaggacccagagaccgggccgctgccgctcgtctcctcttctcgcgggggcacctccaagcccagcagccgcga ggttaaggtgctccccagcgggtggcgggactccggagggcccaccagcaggcgtgcgacgccgccgcggcgttcagttgtttga ggcggggtggacggacgtcctcggcgggagagcggctcgctgggcgccccgagagggcagcagcagcagcgaacaccgaag cgcgtgcgctcggtgcgccaggccgaggcgaggggggcgagattggggccacgaccccgctgttctcggaccgccggcacgtac tggggaggccataattacgacgaggaggtcggtattaaggcgccgggcgtccagaggttcctgccaacaccccaagttttcgcaccc gcctccccgaagagcgctggcgctggccttcgcgttcgctgccttcccgcctggctccagccacgagctccagcttggccgccacgg gcagccgaagactgtacacgcctgggatctgcgcgtgctcctcggggagccgccggggtcccagccccacgccacggagcctcc cgggagcgcgatccacgcggcaggccactaccacgcgcctagtccggctcgtagcgtccagcgcggagctgctccaggcggact cgacccggcggtcgacggctgcggcggggtactgcagcctggcgacgtccgcggccgcttggcgaacaccccgtgcctggtcatc cagatcaccacgcccagagtcagtggggcgcccccaccgccgccctaatctggggggggttggcggccgcagcagcacgagcac ctgtacgtacggtggctgcgatccaacatacaattgacccccgggtcactcccacgtcttctccgtgtacgacggccggctgcgtcgct caactccatccggacactaacccccccaaggccacgtcgccgagtacagatcctcgtacctcgtctagccggccccgcggggccgc ggcgctgctgttcctcctttggggggcagccgaggggttttcctgggggcgccggtccagtaccgatctggaggccgcagcggtctc ccggccaccaaactggcactttatacacagatgtcagctacgcggggtgggcgctcctgatgtgcggggttgtcgtggatccccggg ggtgtagggggtggccggtgccggtgatgttaacggtgcgcacgcgtatgcaatttacgacgaggtgggagggcagcgcgttaaag gggaaaccatcccgggtggcggcggggccggacctccgcacctggtgatgcactggccgcgtttgaccgcgggccggggtcccg cggggttcattccacagggctcgaggcgggccggtggtcggcgccgtcacgtcgtgcgcctcccagcagcgccgggggcgtcttta cgtgccacgcgcgctcgaacgggcgcggggccgccggggtgagtcccagtcgtcacaggaccctctttttaatatctatcgccgagc gggaggagatgccccagggctaagaggtccattgggtacgggaccaaagcgccggcggcgcaccccgcgcggagatcgactag cggcggcgcctctgcggtcgccaggggactccagaatcgcgcctcacgacacccacccctatgacctgttggcgcgaatctccacat tggagacgtccgcggagcgggcacagctcgcccccactcccccgtatacggactccatctttcggcatcgcgcaaggggcaggctg gaagactgtgcgttcgcggggcgtgaggatggcgcgcctctagctcgctgttcgcatctttcctgtgcggcgcaccccgaccgcggc ggcgcctctccgcggcggcggcggctcccaggcctccgacccggtcctcacgcctggcagcacgcggaggtgtccttgaacgga gtactactgcaccgcgttgtcgccgccgacgccggtgtgactgacggcctcctcgcggcgtcgtcgtcggggcgttccatccgggtat ctcgcttcccccggcggagtcgtcgtccaggcctccgcgtcatgccgcgcgaaatcgccggcacgccatggacccctcttcttcccc gcccgacggcccggtgcgtgcccgccagcgccggcacgccatgacccctcttcttcccccgcccgacggcccgcgtgcgtgcccg ccaccagcagagccagctctggagtttgtgcaatccaggtcgggcgcagtactccttaaccggggcgcgaactggtttgtgtgtgcgg ggtgtcgcctgcggcgaccaggctaaggccctaccgggacctcgaggggtcggcgaccgccaaaggggccgcgggtactccag gatgatgtctgcttttacgccacgccctcctccgcgctgttgacaaacagggccgaattttaacacagaagcgccaccagacggtgac cgtatccacggccagtacgagcttcttcggggtgaatgttggcggcgatgtcgcggacatcccccaaagcgagctccgaccacgctt ggggcgccaaacacgttatacagcgtgttcgaattcgcaggcccagcgcacagtaacgtggaccaccgttggcctcgtcaaagcgc cgggcgggtacgccacctccagggcgaccgggccaacccaatccggtctatgagcagcagcagctcgtcggctgacgcggccgtt gcgcctttcagtttacaaccacccgcacctacgaccggtgcgtttagcgtgcccggtgcgcggggagactgtttccagggggtcggg acgccagcgagtcaagctcagcatccttaacgatagacgtacttaactcgcttctggttaaagccgcgtctgggcagcccggaggtca cctcgcgggcctcgtgcagtcgtacaggggaagcgcctcgtgctcgcgccatcgccacgtcgatccaaggagttctcgcgcccgcc ctcccccaatcccccccgagggccgagccggtttcgacgcacccccccaggactggaagaccccctggcaggccgaaaacagca ggcgccactgccctggtcgaacaccgaggcccagaccgccgctgcggcccgtatccgatacagccttgcacccaacacccccaaa ccttccccgctgaagcccccaggctttgccgtccccctgaccgaccctccggtatcaaactcttttacggggcgcggccccgcaggc gtgcctgggcgatttccgacgaagcaccagaaagcacaaaccgcgccctccccgtctgacgatctgcacccccgacgagggttggc cgtcgcgtggcccgagcggcccgtgcacacctggccgtggataaggcggtcgcagtggtccgtctcgctccgccgtctggctgccg tgggcctctgaccagttggctgtggttaactgcggtttaaggacggggtcagggggtgctgtgagattcgcgctcgaccaccaccaag cttggccctcgaacttcgtgccgggccaccgaactgacaaaaaattgacgcccacaacccggtccggaggtcgtgaggagccagaa cgctccggtcacgttccgggaccgctggagcagcgccggtgtcccataatacagcgcggttcgccagctcgctcagcgagcgctgg ccgtctggtccagacaggcggcagcgcgcagctcgcccgcaagcgccaggggggacgggggttggggttggggctgcgccatta ccacgcgggtgggggcgggagcaatacaccgccgggacgaggccgtggccaggaactgggggcccgcgaggagggcaggac ccagagcgaccgctgccgacccagtcctcttctccccttgcacctcccccttagcagccgctgcaggtgctcgcgaattgggggtggc ggaggcgcgagggccgtcgggcaggcgtggtacgtgcgccgcggcgcagtattgagacggacccggggacggacggccccgt cggcgggagagtactgggcggcttggcggatgggcaggtagcgcgtgcgcacgcggtagccaggcccagaggcgggggctgg gttggtggccacgggatttactgttctcgttcctccggcacgtacctgcaggccaagccgccaggatgacaatgacttaaggcgtcga cgccagagggtagagccggtgtagtggcagtacggtgccgcgcgggtgttaacggtcagcgagtatgcagtttaggggaggtggc ggacgacgcagcgcgttggggaggaaaccatctccttgcggcgggtgacgtcctccgcacacattcgatgcactggaccacgtgac cgcggagcggcgtcccgcggaaactccattccacagtacctgcgggccgcgcgggtcggcgccgtggccgcgcctccagcaggg ggaaaacccactgcctgctgcgcctgctcgaacgcccccgccgccggagtcgtcaggatgtcccagtgggtggaccctgttcgacc ctatatagtttggtggaggaaccggcgatctagggcggagcgcccattgggtggggttcaaagcgcttcgacctgacgcccgccccc gactacgcgaccctggcgccgcggggggcgacggggaacagcggaatcgcgcctcacctcccaacgcctcccctgttcacattgc cccgccgccgcggagccgtagagctcgcccccgagcccactcccccgtatctctttgtgctgtgccgatcgcgcaaggcggcgctg gaagactgccagtgcggggcccgtccctcgcggctcaactggctcgctgtcgtcatccttcctgtgtgtagttcccgaccacggcgcg cctctgcgtggggggccgcggagctcgcctccgattcgtccgcctcacgccccctacgcggatcgggcgatacctcctctcatacctc catctcgtcgccgcggatcagggagtgggcggaggcctcctcggcctccatcggcgtcggcgtggcgggaccaaaaagcttcccc ccgggagatcgtcgtcgtcaggtgcgtccggggtgtacacgcgcgaaatccctcgccagctcgccgtccttcttccctcttctcccgcg cctcgcgtggcccgccagcgcctggagccagctgtcgcggtgcaagaacttctcgtgcgggggggcttgcggcgacctcggcgca ggccctgggagcgcctcgaggcggactggcgccagccgcggtttggtatagatcgacgggagtcagcgttgggacgccacggcg agctccgcgctggtgcggtacagggctgggtgcaaacacaggtcgcgcgcaccagaccgtggtcgtatccacgggagggacgatt gttaagccagctttttgtacaacgggtgaatgagctggtgtcggccgacgtaaatcccccaaaacacgagctccgacaacgcgggcg ccctccgtcgggttatacaggtcgcccgaattcgccaatgaagcgcacagcggctgggaccaccgcgctggtcgtcaaagtcgccg gggcgggtaacggtactccagggccgcgtcgccaacccagcgccactatgagccggtagtagctcgtcgggatggcgcggccgtc tggtttttcagtttcatggcccgcacccccagggccggtgcgttgcacgtaactggttccgagcagactgtttccggcgtgtcgggacc ccggagagtcaatggcgtgatccttaaccgctggctgtacttaatgtagcgctggttagggcgaccgtctggggcgttcggaggtcac ccccacagcctcgtgggcacttcaggggattttccgccgtgctcggcccccagccacgtcctcgccgaggagttcgttgaggcgccc tcctcgccggccccccgaccgggttgccggtttatatattcccccccaacgggggaagacccccacggagggccgaaacggccatc gccacaccacgccagtcgaacagcatgttccagaccaccaggtgcggcccgtggccgagcagccttggagtctgccctacacgca caaaccttcccacaccaccccccaactgaccccgtccccctcgtagtaccctccgggggtccactctttgtggacggcggcccgacc cagaccgacgaacgcggacgcacaggccatcaacacgatcttgattggcgacgaggtgcgcgtgcgtcgcgtgccgcctcggccc gtcaagcttggccgtgccccccctcggtcgcaggcgtcccgtctcgctaacggactggctgcggcccgccctctgaccaggcctctg tggttagggcggtaaggagtgcgcgcccagggggtgcgtcacagattcgcgggccgcaccaccaacagcggccctcgaacacgg gcggggccacctggagctaaaaaatcagaggccacaacccgcgcacgaggtcggcgttggtccagaacggcgtcggcactctgg ggcgcgctcacgttcggggatcgctggagcctggtggtgtcccacggtgcacgcgcggtgttgggggtcgctcacgttgttggcatg gccgcggccccagacagaagggcaggcgcagctggggcccgcaagcgggaaagagggggacggttttggggttggggctggg gtcccggtgccgaccaaccattacctctcggatgggggcgcgcctggtgacaccgcgcccctaggccgtgcacacgaactggggg tagctcaggagggcgtgtcgtcgagcgacccggggttgacccagcggctgatctccccttccctcccccctgtgcgtgtaactgcag gacgagcccgaatgtggtttagctgaggcgcccgcgcagccgtcgggcgcgtcttggtacgtgcgggccgcgcagtatgatcagga cccggtcaccttccggccccggcgccccggagtacacgataaacttggcgtgagcggcgtagcgcgtgaggcccaagaaggcgg gggccgcggtagcgcacgtgaggcggagcagcgcgttggtctcggtgcagggatttaccgcggagttcctcactgcctcgtacctg cggctcgtagccgccgaagggaccaatgacgagggcgagtcgacgcagacgcatagagccctgagtcacaggaaacagaaacg cgtcgcccctctccagctcttggaggattctcgactccacgcgcgttttcctccgcagttccaggcgactgcgtgctctccggtgacgtt catcgtcggcctggcactccggccgcaggcaggggcgggcggttggaccacgcggagcaagctgcgccagccgagttctcgtag gagagcgcggtgctgtaccggagtgcagtggtggcgttgtgaaacaggtaggccggtacacacccgttgtcggcccgccttccgtg gcttttttcgagctgacccagcgtccgtggcctagggcgaactcgatggaggcttgcgtggcgatgtggcactgactcaggatatcctg tctgctctcccgccggccgcagcaggcatttggcacacccagtgcgttccggcgcgggacccgaggtcgccctcgatcgccctggc gtcctccagttccgcctttgcagagccaaggaagtggccctccgcgatcggtaacgcgaccaagcgggggcgctggggtttggggt ccgagggcgtgacgtcccagctgggggcggcgcgctcagtaacggccgtgggcgcgaagactcatcgcgcggaacgagctgac gccggtccgggtttccgctttatcctctcgggcatcgtttcccgccgcgaacccgacccgcgcctcgcccgcccaggcggcgccggt tcggaccaccagctcctaagcgccccgtcgatccaggaccaacgccgctaccgatgttccagaacaaacgctgctccgctaaaacct ggggtcacgcgggcagtaaaacacgtccgcgggtcgttaacatcagcgaagcggcggtttagggtcagcagcgacgaccaaaccg actggggatcaccgacgctccttctcgtttccgacgtcctgcactgccattcgctcctatcacacgagccgggccagcgcggcgggc gcggcgaaactcggccgtggtacctgtcggcggttgcgcggggtcgactcgtggcccgggccttgagcagggcgtcggccaccca cacagcatgggcctggccgaccagacccgccttccagctggtcgtcagacgcgagaacttctcccgggcggggtctgaccatacat cgaccgggacaccatttggtcccccctctccggcgattggagggaagcgcacggactcgggggttgatacgggacttcgaaactac ccccgcccccgcgtattgcccttggtgtcggggggggcctgtggggagagcgggtggtccgtgcctcgcgctaccgctcctgttggt ataggtcgtgcctggcgggcggccgccgcttgatcatgtagaaatcccgcaggagccccacgcaaggtgccccagctgcctgtgcc gcggcctcacgggcgtccggcccgcgccagtgccgacgaacgtcccttcaaccactaactggccgccgggcatccccgcggaac actgtagttgtttatgtggcgcgctgggcagggggggggcgcggggcggagctcggcaggcgccgtcttctggtaagcccttttattg atctggcgtctggagcggcatctcccggaaggctctccaaggcgaccaatcaggttggcaggcgggcggaggacccggcctccag cgggtctgcgtggcgggcggcgaaagctgggggcgaagagatccgtgtcctgaggtggtcggcgtcggtgtacagcagccgccc ctcgggcgatttgccgtccgttccctgtcttctcccgccctccgcgtgcgtggtcggcgcctgcaggcgacgtcgcggcgagacggc aatagttattcggagtcgtcaccacgccgccgccctccttcggggtgccgctgtcgggacaggccacgtacgccagcgaggcgacc acccggctcgcgcgcgtcgacgtcgactccccgcctccgggagcctgtgggtccggcaggcgggtgaactccacggccgaggga atggacgccttgctgggttgcggaggtggcgccggatgggacgctccacgactgctgcccgcaccgtcccccgacgacaggggac ggcctcctgaaccgcagacgagcttctcaactgggtcagccaggcgacgaggggtcgctggcgcaggtgagagcccgtccggact gaaggtagattgccaagtttggtctgtagcaaatgaatgacggttcttggtcgtaatccaggataaattcgaggccacacgcgtcaggg acttttggagtcgccctgcgattttcgagcgccgcgtttgttgggaacttgggtgtgccgatacagccgtcgggggccccaaacgggtc tccccgcccccgccttgtttgaggcaggaccacgagcacaaacgtgcgagatcgagtggcgtctaacccgactgcgcgctgcggtc gtccctctacgttgcggggtagcgtccgggcgcttggcagcgcccggcgggtccgctgtcctcgcggtccgcctggcgtcgcggga cagggcctgcttccggccccagcgcagccacccaaagtgccccgaccccgagagtgggggggcctgggtccgcgtcctgcaggg cgccgaactgacagccaccgggccgtgtcacgatcaccgcgggagcggtgcggcctgggttggcgtcgggtccttcgcgtttaagc ttggtctcccatcagcagtttccaatcccggccccggggaatcagaagcggagcgggctggggacggggacggcgtacgagaagg gccagggcgaggtgcggcgcgctttagcgaccgacgcggcaccaacaacgacaccgagccggcggagagccgccgccctcac caacctgttcttacgtcgccggtaggggcagacgccccctgctccccggaccccggcgccggaaccgagccccgcgggggcgtg gccaagccccccgcccctgggcacgcgcacggcgatcagagggtcagtggtgtttcctcatggccccttatcgggggttcccaccaa acctgcggccaacacaacggtggatgcctctctcaagatggg SEQ.3: 15kb Stuffer

[0164] GC Content: 68% ggtcgggcttggatttaatactaacgcgtaaatagtgagctttcacgcgcctccgcctgggagcgcagtctcggtggggatggcctctg tccggcgccgtcgccaggccggtcacgacgccctttttagaatgcaaggaccgtttactgagccttcgctcccgtctccacatagtgat aaaggcacacagcagacgaccgcgtaaggccgtggacgtagcgtttggccaagacgtcccctgggaccgcgccggagacggcg gcggacggccctgcctccggtaccgtatctccgcggacagtcggagtcgtagagttggctgactactacctcgccccagtcgacgaa ccggtacccctcgttcccgcaggccagacgcgcgctacgtcgccctactctccctccataacaccaataactctcagtaacaggcgcg tccggcttctgtgcccgatctctcgccgcccggggcccctctccagtgtcccgacattatccacggcccccgctcgtttgccccgctgc tcggtcgttatgtcggcccccacatcgggggtgatcattgccgcgcccgggggggaaaagcgagcagccggcgcccagatcgaaa tcgtttttgccctcgcagacctttgggtctcgcacctgaggggtgccccaaggtcgatcgccctcctctgaaaatgaaccagaacagca gcggggagtaacccacgtggccccgaccaccacaatcgccaggctgcggaagtcgggcccacgagccgccctccacggccccc gcccgccccctctcggcgatgtgggttgctcgtggacgcgtgcctggacc aacccc aactcc agcccc agtgtccccttttcgcggc a aagactggttgtccgtggccgaggacgtttcgttgcggcgaggtggacgttacgaacacgtcgatatatattaaaggtgcaggatgtgt tttcgtgggcctcttccagtataagggttggtcgcctgcggtcctgacggccaagcgtccaaccgtttattcgccccagattatcttcccg ctacgaccgcgcggcggagcaccgcctggttatacccgtgacgcccccgagttggtaccgtatgcgtgcgaattggtcggggccaa agcgggggagcggttttgaggtccagcgaattgagcgtgaagtttaccccggtatgggtccggcggcgccgggcgcgccgaccac gctagagagaacgtggagccactcaccggcgtgtccggacaactacggcaccacccacaagtgtacgacgacgcagccgttccga cgcctcaagcacgcccccgcgcgccagcctgtttgtcccccgaccccagcctacgtgggccgcccaggtgcgcaagcgctgagtta ccccgcgtgtccattgcgtcctcgggcgacttcaaatgccccgggttttttgagcacgtccccattcggcacgtcccctcgagggtggg gggcaggccccacctgggggcccccgcccgaacatcagggggtgcggttaacacctactttctggccgggggaccaggcggcgg cgggcgcgtcgcctctcttctaccactgtccgccagcatggcggcatggcgggcccgttcccccttccccgcgcacgcggcggagg gctagcgtgggcattttgcttccgctgtaccgcattccgcgtttggtgtatgtggttgtacgcgccgtcgctcccgcccctcgaccgactg cccgggcacccggaggtccgggaggcccgacgaagatgctagcggcctataaaaaaggacgccgcctacgggaaccgcgtgcg catgtgcaggggcgccccaccgcgggaggcctcctttgccaaacacaaccggtgccgtcgtcgaccgcttcggggcctcttcggca tgtggtctgccgaaccaagccttgaggggccggattggggggccggaagcgtaggctgaagacggggtgtcgccgagggcggtc ctgcacgagccagtactcgggcgtctgcgggcaggtcgcacgacgacgcgtggactgggagcgccgacacgtcgatgaaatctcc gatgtgggacgcccgcagagtggcgccgcaaatcacgccccccgtggaccgtctcgcctggccgtctgtcgggcggattggctccc tgggagacgtggtctccggttccgcgacagcgagcgcgtagcgcggttccggtgtgtccccagaagcgacggggagccgatttctc ggctagtatatgcccgagtggaccacgcggtccgcgctcggatgttgttccagtagccgtggaccgtctttcatgtaaagcagctgggt actttttatgtctgtactaccccggggatcgtgagcaggttccgtggtggcctgaaacacagcatcttggccagccagcagatccgcgc gaaggccacatacgcggggcaaggaggggcgtggccggggtggtgacggcgagaagaacccccccgttacctttgcgcctccgg gctgcaaacccggccggatgacagctcgtcgaccacccggacaactttcactcgcacgtacgcgcgtcgagcgtggcgtcggggg gcggagcctgacatgagggccggtcacgagttttccccgcacgccccccgtggactcggtgtgggcgtgtactcgtagtagacccc ggcctggacggagaaacagaatcacgcggccggcgtcccatgcctctacggggacacgggatgtcggacgacacgcaggctgga gacggaccagcaacgcgctgcttccagtactgcgttctcctccagagcctaaaaaacgtttgtattgtccgccgaagcgggcccgtca cgggggggtcaaacagcaggtccccgatcgccgttacggttctggccgcctccggccgaaaacgctggaccgcgaggatggtgga ctaaaaacagggcgctcgggaacgccctttccctgctacgacacggccgtgcccgccctgggcgcggcgccccgtactgcagtgg cggccccgcccacctatctgtccgcctcggaggttctggagccacccggcgcgaagctgctccgctgtttacttgccgtcctttggatc cccgcgctcgacggacccgggggttgtagccctcctctaccaggagggcgcaggaccccccggcgcttgggaaggggccccccg gtaaaataccgacccccccggcgttggggttttgtgggggtgtcgcctgcgaccttgtccaggctgccaccacttaccgggtgcgcca ggtcggcggggagcacaaggggagcgccgcctccaggggtttccctgcttttgaacgttacgccctcgtctcggccgagttgctcctc cacgcgaatgcgacccccaagcgcgcctgcagcagggtgctcgccacggcgatccgggaaggccagtaagggccggggcttcg caggcagcgatgtttctcccaaaaacacggcaactatgtacgaaggctcgaaaacacgctgcccaggcaaacacgctgcatctgcgt gttgtggtcgtcctacagactcccgattaacgttcggctagcttggccgtgagatatacgccgcggcctcgtgcgccacaacagaagc cgaccgcggtgagcatccggcttgtggagcagcagacggcgcgcatgacgcctccaggtgcgcctggataacaaccacccgcca gggcgctacgacataggcgcggggagagggctccccaggggaccacgtgtactgcctgcgctccctcagcagccgataagcgac cttctggactcgccgtacgcgaagccggggtcagccgccagcgtgccctcgcgggccatcccccccagtcgttgtcgcccagcgcc ttcggcagcgcctccggaggtggtgagccgatccctcgcgccctcggtcaccccaactctactgggggccgctgggggccccgac gcggacgaggactggcctcaacgccacctgccaaatacagcggcccactagggtgcccaggcgggcggccgagagcacggtgc cgctgggtagaatccgatacgacctcgggcagcccccggagacccccaggtcgtccctcgcatccccctggggggcttctggacca gctgaccgtctggtatcaaactccggggcctacggggagctaatcgcgatttcagacagcaccagaagtgccgctcccgtcttgcacc cccgacgaacgttggccgtcgcgggcggcccgagcggccgtgcacagcctggcccaagataaggcggtccaccgcgtggtccgg acgcgggccgccgtcccgaggcgtgggccggtgtgaaagttggcggcgcgactgcggtttgcacaggagcggggcaggcgctgt gagatccgcccctcgaccaccacgtccgtcgcttgccagtttcgtgccgggtatgccgaactgacgccacttgacgcccagccagcg cggtccggcccgatatgaggagcgaccgaaaaggtcacgaccgcgctccggtctgatgctgcgggaccgtgaagtagcgccggc gctgtaatacagcgtacatcgccagctcggagatctctgcgagcgccacggtctggtccgagctgctcgcccgcggtttccgggcca ggggggttggggttggggtcctcgggcgcagccgcgggacgcgggtgcccgtggagcaagatcagccgggacgagaagaggg ccaggaacttgctgcccgcgaggccggaggacccagagaccgggccgctgccgctcgtctcctcttctcgcgggggcacctccaa gcccagcagccgcgaggttaaggtgctccccagcgggtggcgggactccggagggcccaccagcaggcgtgcgacgccgccgc ggcgttcagttgtttgaggcggggtggacggacgtcctcggcgggagagcggctcgctgggcgccccgagagggcagcagcagc agcgaacaccgaagcgcgtgcgctcggtgcgccaggccgaggcgaggggggcgagattggggccacgaccccgctgttctcgg accgccggcacgtactggggaggccataattacgacgaggaggtcggtattaaggcgccgggcgtccagaggttcctgccaacac cccaagttttcgcacccgcctccccgaagagcgctggcgctggccttcgcgttcgctgccttcccgcctggctccagccacgagctcc agcttggccgccacgggcagccgaagactgtacacgcctgggatctgcgcgtgctcctcggggagccgccggggtcccagcccca cgccacggagcctcccgggagcgcgatccacgcggcaggccactaccacgcgcctagtccggctcgtagcgtccagcgcggagc tgctccaggcggactcgacccggcggtcgacggctgcggcggggtactgcagcctggcgacgtccgcggccgcttggcgaacac cccgtgcctggtcatccagatcaccacgcccagagtcagtggggcgcccccaccgccgccctaatctggggggggttggcggccg cagcagcacgagcacctgtacgtacggtggctgcgatccaacatacaattgacccccgggtcactcccacgtcttctccgtgtacgac ggccggctgcgtcgctcaactccatccggacactaacccccccaaggccacgtcgccgagtacagatcctcgtacctcgtctagccg gccccgcggggccgcggcgctgctgttcctcctttggggggcagccgaggggttttcctgggggcgccggtccagtaccgatctgg aggccgcagcggtctcccggccaccaaactggcactttatacacagatgtcagctacgcggggtgggcgctcctgatgtgcggggtt gtcgtggatccccgggggtgtagggggtggccggtgccggtgatgttaacggtgcgcacgcgtatgcaatttacgacgaggtggga gggcagcgcgttaaaggggaaaccatcccgggtggcggcggggccggacctccgcacctggtgatgcactggccgcgtttgacc gcgggccggggtcccgcggggttcattccacagggctcgaggcgggccggtggtcggcgccgtcacgtcgtgcgcctcccagca gcgccgggggcgtctttacgtgccacgcgcgctcgaacgggcgcggggccgccggggtgagtcccagtcgtcacaggaccctctt tttaatatctatcgccgagcgggaggagatgccccagggctaagaggtccattgggtacgggaccaaagcgccggcggcgcaccc cgcgcggagatcgactagcggcggcgcctctgcggtcgccaggggactccagaatcgcgcctcacgacacccacccctatgacct gttggcgcgaatctccacattggagacgtccgcggagcgggcacagctcgcccccactcccccgtatacggactccatctttcggcat cgcgcaaggggcaggctggaagactgtgcgttcgcggggcgtgaggatggcgcgcctctagctcgctgttcgcatctttcctgtgcg gcgcaccccgaccgcggcggcgcctctccgcggcggcggcggctcccaggcctccgacccggtcctcacgcctggcagcacgc ggaggtgtccttgaacggagtactactgcaccgcgttgtcgccgccgacgccggtgtgactgacggcctcctcgcggcgtcgtcgtc ggggcgttccatccgggtatctcgcttcccccggcggagtcgtcgtccaggcctccgcgtcatgccgcgcgaaatcgccggcacgc catggacccctcttcttccccgcccgacggcccggtgcgtgcccgccagcgccggcacgccatgacccctcttcttcccccgcccga cggcccgcgtgcgtgcccgccaccagcagagccagctctggagtttgtgcaatccaggtcgggcgcagtactccttaaccggggcg cgaactggtttgtgtgtgcggggtgtcgcctgcggcgaccaggctaaggccctaccgggacctcgaggggtcggcgaccgccaaa ggggccgcgggtactccaggatgatgtctgcttttacgccacgccctcctccgcgctgttgacaaacagggccgaattttaacacaga agcgccaccagacggtgaccgtatccacggccagtacgagcttcttcggggtgaatgttggcggcgatgtcgcggacatcccccaa agcgagctccgaccacgcttggggcgccaaacacgttatacagcgtgttcgaattcgcaggcccagcgcacagtaacgtggaccac cgttggcctcgtcaaagcgccgggcgggtacgccacctccagggcgaccgggccaacccaatccggtctatgagcagcagcagct cgtcggctgacgcggccgttgcgcctttcagtttacaaccacccgcacctacgaccggtgcgtttagcgtgcccggtgcgcggggag actgtttccagggggtcgggacgccagcgagtcaagctcagcatccttaacgatagacgtacttaactcgcttctggttaaagccgcgt ctgggcagcccggaggtcacctcgcgggcctcgtgcagtcgtacaggggaagcgcctcgtgctcgcgccatcgccacgtcgatcc aaggagttctcgcgcccgccctcccccaatcccccccgagggccgagccggtttcgacgcacccccccaggactggaagaccccc tggcaggccgaaaacagcaggcgccactgccctggtcgaacaccgaggcccagaccgccgctgcggcccgtatccgatacagcc ttgcacccaacacccccaaaccttccccgctgaagcccccaggctttgccgtccccctgaccgaccctccggtatcaaactcttttacg gggcgcggccccgcaggcgtgcctgggcgatttccgacgaagcaccagaaagcacaaaccgcgccctccccgtctgacgatctgc acccccgacgagggttggccgtcgcgtggcccgagcggcccgtgcacacctggccgtggataaggcggtcgcagtggtccgtctc gctccgccgtctggctgccgtgggcctctgaccagttggctgtggttaactgcggtttaaggacggggtcagggggtgctgtgagatt cgcgctcgaccaccaccaagcttggccctcgaacttcgtgccgggccaccgaactgacaaaaaattgacgcccacaacccggtccg gaggtcgtgaggagccagaacgctccggtcacgttccgggaccgctggagcagcgccggtgtcccataatacagcgcggttcgcc agctcgctcagcgagcgctggccgtctggtccagacaggcggcagcgcgcagctcgcccgcaagcgccaggggggacgggggt tggggttggggctgcgccattaccacgcgggtgggggcgggagcaatacaccgccgggacgaggccgtggccaggaactgggg gcccgcgaggagggcaggacccagagcgaccgctgccgacccagtcctcttctccccttgcacctcccccttagcagccgctgcag gtgctcgcgaattgggggtggcggaggcgcgagggccgtcgggcaggcgtggtacgtgcgccgcggcgcagtattgagacggac ccggggacggacggccccgtcggcgggagagtactgggcggcttggcggatgggcaggtagcgcgtgcgcacgcggtagccag gcccagaggcgggggctgggttggtggccacgggatttactgttctcgttcctccggcacgtacctgcaggccaagccgccaggatg acaatgacttaaggcgtcgacgccagagggtagagccggtgtagtggcagtacggtgccgcgcgggtgttaacggtcagcgagtat gcagtttaggggaggtggcggacgacgcagcgcgttggggaggaaaccatctccttgcggcgggtgacgtcctccgcacacattcg atgcactggaccacgtgaccgcggagcggcgtcccgcggaaactccattccacagtacctgcgggccgcgcgggtcggcgccgtg gccgcgcctccagcagggggaaaacccactgcctgctgcgcctgctcgaacgcccccgccgccggagtcgtcaggatgtcccagt gggtggaccctgttcgaccctatatagtttggtggaggaaccggcgatctagggcggagcgcccattgggtggggttcaaagcgcttc gacctgacgcccgcccccgactacgcgaccctggcgccgcggggggcgacggggaacagcggaatcgcgcctcacctcccaac gcctcccctgttcacattgccccgccgccgcggagccgtagagctcgcccccgagcccactcccccgtatctctttgtgctgtgccgat cgcgcaaggcggcgctggaagactgccagtgcggggcccgtccctcgcggctcaactggctcgctgtcgtcatccttcctgtgtgta gttcccgaccacggcgcgcctctgcgtggggggccgcggagctcgcctccgattcgtccgcctcacgccccctacgcggatcggg cgatacctcctctcatacctccatctcgtcgccgcggatcagggagtgggcggaggcctcctcggcctccatcggcgtcggcgtggc gggaccaaaaagcttccccccgggagatcgtcgtcgtcaggtgcgtccggggtgtacacgcgcgaaatccctcgccagctcgccgt ccttcttccctcttctcccgcgcctcgcgtggcccgccagcgcctggagccagctgtcgcggtgcaagaacttctcgtgcggggggg cttgcggcgacctcggcgcaggccctgggagcgcctcgaggcggactggcgccagccgcggtttggtatagatcgacgggagtca gcgttgggacgccacggcgagctccgcgctggtgcggtacagggctgggtgcaaacacaggtcgcgcgcaccagaccgtggtcg tatccacgggagggacgattgttaagccagctttttgtacaacgggtgaatgagctggtgtcggccgacgtaaatcccccaaaacacg agctccgacaacgcgggcgccctccgtcgggttatacaggtcgcccgaattcgccaatgaagcgcacagcggctgggaccaccgc gctggtcgtcaaagtcgccggggcgggtaacggtactccagggccgcgtcgccaacccagcgccactatgagccggtagtagctc gtcgggatggcgcggccgtctggtttttcagtttcatggcccgcacccccagggccggtgcgttgcacgtaactggttccgagcagac tgtttccggcgtgtcgggaccccggagagtcaatggcgtgatccttaaccgctggctgtacttaatgtagcgctggttagggcgaccgt ctggggcgttcggaggtcacccccacagcctcgtgggcacttcaggggattttccgccgtgctcggcccccagccacgtcctcgccg aggagttcgttgaggcgccctcctcgccggccccccgaccgggttgccggtttatatattcccccccaacgggggaagacccccacg gagggccgaaacggccatcgccacaccacgccagtcgaacagcatgttccagaccaccaggtgcggcccgtggccgagcagcct tggagtctgccctacacgcacaaaccttcccacaccaccccccaactgaccccgtccccctcgtagtaccctccgggggtccactcttt gtggacggcggcccgacccagaccgacgaacgcggacgcacaggccatcaacacgatcttgattggcgacgaggtgcgcgtgcg tcgcgtgccgcctcggcccgtcaagcttggccgtgccccccctcggtcgcaggcgtcccgtctcgctaacggactggctgcggccc gccctctgaccaggcctctgtggttagggcggtaaggagtgcgcgcccagggggtgcgtcacagattcgcgggccgcaccaccaa cagcggccctcgaacacgggcggggccacctggagctaaaaaatcagaggccacaacccgcgcacgaggtcggcgttggtccag aacggcgtcggcactctggggcgcgctcacgttcggggatcgctggagcctggtggtgtcccacggtgcacgcgcggtgttgggg gtcgctcacgttgttggcatggccgcggccccagacagaagggcaggcgcagctggggcccgcaagcgggaaagagggggacg gttttggggttggggctggggtcccggtgccgaccaaccattacctctcggatgggggcgcgcctggtgacaccgcgcccctaggc cgtgcacacgaactgggggtagctcaggagggcgtgtcgtcgagcgacccggggttgacccagcggctgatctccccttccctccc ccctgtgcgtgtaactgcaggacgagcccgaatgtggtttagctgaggcgcccgcgcagccgtcgggcgcgtcttggtacgtgcgg gccgcgcagtatgatcaggacccggtcaccttccggccccggcgccccggagtacacgataaacttggcgtgagcggcgtagcgc gtgaggcccaagaaggcgggggccgcggtagcgcacgtgaggcggagcagcgcgttggtctcggtgcagggatttaccgcgga gttcctcactgcctcgtacctgcggctcgtagccgccgaagggaccaatgacgagggcgagtcgacgcagacgcatagagccctga gtcacaggaaacagaaacgcgtcgcccctctccagctcttggaggattctcgactccacgcgcgttttcctccgcagttccaggcgact gcgtgctctccggtgacgttcatcgtcggcctggcactccggccgcaggcaggggcgggcggttggaccacgcggagcaagctgc gccagccgagttctcgtaggagagcgcggtgctgtaccggagtgcagtggtggcgttgtgaaacaggtaggccggtacacacccgt tgtcggcccgccttccgtggcttttttcgagctgacccagcgtccgtggcctagggcgaactcgatggaggcttgcgtggcgatgtgg cactgactcaggatatcctgtctgctctcccgccggccgcagcaggcatttggcacacccagtgcgttccggcgcgggacccgaggt cgccctcgatcgccctggcgtcctccagttccgcctttgcagagccaaggaagtggccctccgcgatcggtaacgcgaccaagcgg gggcgctggggtttggggtccgagggcgtgacgtcccagctgggggcggcgcgctcagtaacggccgtgggcgcgaagactcat cgcgcggaacgagctgacgccggtccgggtttccgctttatcctctcgggcatcgtttcccgccgcgaacccgacccgcgcctcgcc cgcccaggcggcgccggttcggaccaccagctcctaagcgccccgtcgatccaggaccaacgccgctaccgatgttccagaacaa acgctgctccgctaaaacctggggtcacgcgggcagtaaaacacgtccgcgggtcgttaacatcagcgaagcggcggtttagggtc agcagcgacgaccaaaccgactggggatcaccgacgctccttctcgtttccgacgtcctgcactgccattcgctcctatcacacgagc cgggccagcgcggcgggcgcggcgaaactcggccgtggtacctgtcggcggttgcgcggggtcgactcgtggcccgggccttga gcagggcgtcggccacccacacagcatgggcctggccgaccagacccgccttccagctggtcgtcagacgcgagaacttctcccg ggcggggtctgaccatacatcgaccgggacaccatttggtcccccctctccggcgattggagggaagcgcacggactcgggggttg atacgggacttcgaaactacccccgcccccgcgtattgcccttggtgtcggggggggcctgtggggagagcgggtggtccgtgcct cgcgctaccgctcctgttggtataggtcgtgcctggcgggcggccgccgcttgatcatgtagaaatcccgcaggagccccacgcaag gtgccccagctgcctgtgccgcggcctcacgggcgtccggcccgcgccagtgccgacgaacgtcccttcaaccactaactggccgc cgggcatccccgcggaacactgtagttgtttatgtggcgcgctgggcagggggggggcgcggggcggagctcggcaggcgccgt cttctggtaagcccttttattgatctggcgtctggagcggcatctcccggaaggctctccaaggcgaccaatcaggttggcaggcgggc ggaggacccggcctccagcgggtctgcgtggcgggcggcgaaagctgggggcgaagagatccgtgtcctgaggtggtcggcgtc ggtgtacagcagccgcccctcgggcgatttgccgtccgttccctgtcttctcccgccctccgcgtgcgtggtcggcgcctgcaggcga cgtcgcggcgagacggcaatagttattcggagtcgtcaccacgccgccgccctccttcggggtgccgctgtcgggacaggccacgt acgccagcgaggcgaccacccggctcgcgcgcgtcgacgtcgactccccgcctccgggagcctgtgggtccggcaggcgggtga actccacggccgagggaatggacgccttgctgggttgcggaggtggcgccggatgggacgctccacgactgctgcccgcaccgtc ccccgacgacaggggacggcctcctgaaccgcagacgagcttctcaactgggtcagccaggcgacgaggggtcgctggcgcagg tgagagcccgtccggactgaaggtagattgccaagtttggtctgtagcaaatgaatgacggttcttggtcgtaatccaggataaattcga ggccacacgcgtcagggacttttggagtcgccctgcgattttcgagcgccgcgtttgttgggaacttgggtgtgccgatacagccgtcg ggggccccaaacgggtctccccgcccccgccttgtttgaggcaggaccacgagcacaaacgtgcgagatcgagtggcgtctaacc cgactgcgcgctgcggtcgtccctctacgttgcggggtagcgtccgggcgcttggcagcgcccggcgggtccgctgtcctcgcggt ccgcctggcgtcgcgggacagggcctgcttccggccccagcgcagccacccaaagtgccccgaccccgagagtgggggggcct gggtccgcgtcctgcagggcgccgaactgacagccaccgggccgtgtcacgatcaccgcgggagcggtgcggcctgggttggcg tcgggtccttcgcgtttaagcttggtctcccatcagcagtttccaatcccggccccggggaatcagaagcggagcgggctggggacg gggacggcgtacgagaagggccagggcgaggtgcggcgcgctttagcgaccgacgcggcaccaacaacgacaccgagccggc ggagagccgccgccctcaccaacctgttcttacgtcgccggtaggggcagacgccccctgctccccggaccccggcgccggaacc gagccccgcgggggcgtggccaagccccccgcccctgggcacgcgcacggcgatcagagggtcagtggtgtttcctcatggccc cttatcgggggttcccaccaaacctgcggccaacacaacggtggatgcctctctcaagatggg

[0165] SEQ.4: 12kb Staffer

[0166] GC content: 68% ggtcgggcttggatttaatactaacgcgtaaatagtgagctttcacgcgcctccgcctgggagcgcagtctcggtggggatggcctctg tccggcgccgtcgccaggccggtcacgacgccctttttagaatgcaaggaccgtttactgagccttcgctcccgtctccacatagtgat aaaggcacacagcagacgaccgcgtaaggccgtggacgtagcgtttggccaagacgtcccctgggaccgcgccggagacggcg gcggacggccctgcctccggtaccgtatctccgcggacagtcggagtcgtagagttggctgactactacctcgccccagtcgacgaa ccggtacccctcgttcccgcaggccagacgcgcgctacgtcgccctactctccctccataacaccaataactctcagtaacaggcgcg tccggcttctgtgcccgatctctcgccgcccggggcccctctccagtgtcccgacattatccacggcccccgctcgtttgccccgctgc tcggtcgttatgtcggcccccacatcgggggtgatcattgccgcgcccgggggggaaaagcgagcagccggcgcccagatcgaaa tcgtttttgccctcgcagacctttgggtctcgcacctgaggggtgccccaaggtcgatcgccctcctctgaaaatgaaccagaacagca gcggggagtaacccacgtggccccgaccaccacaatcgccaggctgcggaagtcgggcccacgagccgccctccacggccccc gcccgccccctctcggcgatgtgggttgctcgtggacgcgtgcctggacc aacccc aactcc agcccc agtgtccccttttcgcggc a aagactggttgtccgtggccgaggacgtttcgttgcggcgaggtggacgttacgaacacgtcgatatatattaaaggtgcaggatgtgt tttcgtgggcctcttccagtataagggttggtcgcctgcggtcctgacggccaagcgtccaaccgtttattcgccccagattatcttcccg ctacgaccgcgcggcggagcaccgcctggttatacccgtgacgcccccgagttggtaccgtatgcgtgcgaattggtcggggccaa agcgggggagcggttttgaggtccagcgaattgagcgtgaagtttaccccggtaatttcagacagcaccagaagtgccgctcccgtct tgcacccccgacgaacgttggccgtcgcgggcggcccgagcggccgtgcacagcctggcccaagataaggcggtccaccgcgtg gtccggacgcgggccgccgtcccgaggcgtgggccggtgtgaaagttggcggcgcgactgcggtttgcacaggagcggggcag gcgctgtgagatccgcccctcgaccaccacgtccgtcgcttgccagtttcgtgccgggtatgccgaactgacgccacttgacgcccag ccagcgcggtccggcccgatatgaggagcgaccgaaaaggtcacgaccgcgctccggtctgatgctgcgggaccgtgaagtagc gccggcgctgtaatacagcgtacatcgccagctcggagatctctgcgagcgccacggtctggtccgagctgctcgcccgcggtttcc gggccaggggggttggggttggggtcctcgggcgcagccgcgggacgcgggtgcccgtggagcaagatcagccgggacgaga agagggccaggaacttgctgcccgcgaggccggaggacccagagaccgggccgctgccgctcgtctcctcttctcgcgggggca cctccaagcccagcagccgcgaggttaaggtgctccccagcgggtggcgggactccggagggcccaccagcaggcgtgcgacg ccgccgcggcgttcagttgtttgaggcggggtggacggacgtcctcggcgggagagcggctcgctgggcgccccgagagggcag cagcagcagcgaacaccgaagcgcgtgcgctcggtgcgccaggccgaggcgaggggggcgagattggggccacgaccccgct gttctcggaccgccggcacgtactggggaggccataattacgacgaggaggtcggtattaaggcgccgggcgtccagaggttcctg ccaacaccccaagttttcgcacccgcctccccgaagagcgctggcgctggccttcgcgttcgctgccttcccgcctggctccagccac gagctccagcttggccgccacgggcagccgaagactgtacacgcctgggatctgcgcgtgctcctcggggagccgccggggtccc agccccacgccacggagcctcccgggagcgcgatccacgcggcaggccactaccacgcgcctagtccggctcgtagcgtccagc gcggagctgctccaggcggactcgacccggcggtcgacggctgcggcggggtactgcagcctggcgacgtccgcggccgcttgg cgaacaccccgtgcctggtcatccagatcaccacgcccagagtcagtggggcgcccccaccgccgccctaatctggggggggttg gcggccgcagcagcacgagcacctgtacgtacggtggctgcgatccaacatacaattgacccccgggtcactcccacgtcttctccg tgtacgacggccggctgcgtcgctcaactccatccggacactaacccccccaaggccacgtcgccgagtacagatcctcgtacctcg tctagccggccccgcggggccgcggcgctgctgttcctcctttggggggcagccgaggggttttcctgggggcgccggtccagtac cgatctggaggccgcagcggtctcccggccaccaaactggcactttatacacagatgtcagctacgcggggtgggcgctcctgatgt gcggggttgtcgtggatccccgggggtgtagggggtggccggtgccggtgatgttaacggtgcgcacgcgtatgcaatttacgacga ggtgggagggcagcgcgttaaaggggaaaccatcccgggtggcggcggggccggacctccgcacctggtgatgcactggccgc gtttgaccgcgggccggggtcccgcggggttcattccacagggctcgaggcgggccggtggtcggcgccgtcacgtcgtgcgcct cccagcagcgccgggggcgtctttacgtgccacgcgcgctcgaacgggcgcggggccgccggggtgagtcccagtcgtcacagg accctctttttaatatctatcgccgagcgggaggagatgccccagggctaagaggtccattgggtacgggaccaaagcgccggcggc gcaccccgcgcggagatcgactagcggcggcgcctctgcggtcgccaggggactccagaatcgcgcctcacgacacccacccct atgacctgttggcgcgaatctccacattggagacgtccgcggagcgggcacagctcgcccccactcccccgtatacggactccatctt tcggcatcgcgcaaggggcaggctggaagactgtgcgttcgcggggcgtgaggatggcgcgcctctagctcgctgttcgcatctttc ctgtgcggcgcaccccgaccgcggcggcgcctctccgcggcggcggcggctcccaggcctccgacccggtcctcacgcctggca gcacgcggaggtgtccttgaacggagtactactgcaccgcgttgtcgccgccgacgccggtgtgactgacggcctcctcgcggcgt cgtcgtcggggcgttccatccgggtatctcgcttcccccggcggagtcgtcgtccaggcctccgcgtcatgccgcgcgaaatcgccg gcacgccatggacccctcttcttccccgcccgacggcccggtgcgtgcccgccagcgccggcacgccatgacccctcttcttccccc gcccgacggcccgcgtgcgtgcccgccaccagcagagccagctctggagtttgtgcaatccaggtcgggcgcagtactccttaacc ggggcgcgaactggtttgtgtgtgcggggtgtcgcctgcggcgaccaggctaaggccctaccgggacctcgaggggtcggcgacc gccaaaggggccgcgggtactccaggatgatgtctgcttttacgccacgccctcctccgcgctgttgacaaacagggccgaattttaa cacagaagcgccaccagacggtgaccgtatccacggccagtacgagcttcttcggggtgaatgttggcggcgatgtcgcggacatc ccccaaagcgagctccgaccacgcttggggcgccaaacacgttatacagcgtgttcgaattcgcaggcccagcgcacagtaacgtg gaccaccgttggcctcgtcaaagcgccgggcgggtacgccacctccagggcgaccgggccaacccaatccggtctatgagcagca gcagctcgtcggctgacgcggccgttgcgcctttcagtttacaaccacccgcacctacgaccggtgcgtttagcgtgcccggtgcgcg gggagactgtttccagggggtcgggacgccagcgagtcaagctcagcatccttaacgatagacgtacttaactcgcttctggttaaag ccgcgtctgggcagcccggaggtcacctcgcgggcctcgtgcagtcgtacaggggaagcgcctcgtgctcgcgccatcgccacgt cgatccaaggagttctcgcgcccgccctcccccaatcccccccgagggccgagccggtttcgacgcacccccccaggactggaag accccctggcaggccgaaaacagcaggcgccactgccctggtcgaacaccgaggcccagaccgccgctgcggcccgtatccgat acagccttgcacccaacacccccaaaccttccccgctgaagcccccaggctttgccgtccccctgaccgaccctccggtatcaaactc ttttacggggcgcggccccgcaggcgtgcctgggcgatttccgacgaagcaccagaaagcacaaaccgcgccctccccgtctgac gatctgcacccccgacgagggttggccgtcgcgtggcccgagcggcccgtgcacacctggccgtggataaggcggtcgcagtggt ccgtctcgctccgccgtctggctgccgtgggcctctgaccagttggctgtggttaactgcggtttaaggacggggtcagggggtgctgt gagattcgcgctcgaccaccaccaagcttggccctcgaacttcgtgccgggccaccgaactgacaaaaaattgacgcccacaaccc ggtccggaggtcgtgaggagccagaacgctccggtcacgttccgggaccgctggagcagcgccggtgtcccataatacagcgcgg ttcgccagctcgctcagcgagcgctggccgtctggtccagacaggcggcagcgcgcagctcgcccgcaagcgccaggggggacg ggggttggggttggggctgcgccattaccacgcgggtgggggcgggagcaatacaccgccgggacgaggccgtggccaggaac tgggggcccgcgaggagggcaggacccagagcgaccgctgccgacccagtcctcttctccccttgcacctcccccttagcagccgc tgcaggtgctcgcgaattgggggtggcggaggcgcgagggccgtcgggcaggcgtggtacgtgcgccgcggcgcagtattgaga cggacccggggacggacggccccgtcggcgggagagtactgggcggcttggcggatgggcaggtagcgcgtgcgcacgcggta gccaggcccagaggcgggggctgggttggtggccacgggatttactgttctcgttcctccggcacgtacctgcaggccaagccgcc aggatgacaatgacttaaggcgtcgacgccagagggtagagccggtgtagtggcagtacggtgccgcgcgggtgttaacggtcagc gagtatgcagtttaggggaggtggcggacgacgcagcgcgttggggaggaaaccatctccttgcggcgggtgacgtcctccgcaca cattcgatgcactggaccacgtgaccgcggagcggcgtcccgcggaaactccattccacagtacctgcgggccgcgcgggtcggc gccgtggccgcgcctccagcagggggaaaacccactgcctgctgcgcctgctcgaacgcccccgccgccggagtcgtcaggatgt cccagtgggtggaccctgttcgaccctatatagtttggtggaggaaccggcgatctagggcggagcgcccattgggtggggttcaaa gcgcttcgacctgacgcccgcccccgactacgcgaccctggcgccgcggggggcgacggggaacagcggaatcgcgcctcacct cccaacgcctcccctgttcacattgccccgccgccgcggagccgtagagctcgcccccgagcccactcccccgtatctctttgtgctgt gccgatcgcgcaaggcggcgctggaagactgccagtgcggggcccgtccctcgcggctcaactggctcgctgtcgtcatccttcctg tgtgtagttcccgaccacggcgcgcctctgcgtggggggccgcggagctcgcctccgattcgtccgcctcacgccccctacgcggat cgggcgatacctcctctcatacctccatctcgtcgccgcggatcagggagtgggcggaggcctcctcggcctccatcggcgtcggcg tggcgggaccaaaaagcttccccccgggagatcgtcgtcgtcaggtgcgtccggggtgtacacgcgcgaaatccctcgccagctcg ccgtccttcttccctcttctcccgcgcctcgcgtggcccgccagcgcctggagccagctgtcgcggtgcaagaacttctcgtgcgggg gggcttgcggcgacctcggcgcaggccctgggagcgcctcgaggcggactggcgccagccgcggtttggtatagatcgacggga gtcagcgttgggacgccacggcgagctccgcgctggtgcggtacagggctgggtgcaaacacaggtcgcgcgcaccagaccgtg gtcgtatccacgggagggacgattgttaagccagctttttgtacaacgggtgaatgagctggtgtcggccgacgtaaatcccccaaaa cacgagctccgacaacgcgggcgccctccgtcgggttatacaggtcgcccgaattcgccaatgaagcgcacagcggctgggacca ccgcgctggtcgtcaaagtcgccggggcgggtaacggtactccagggccgcgtcgccaacccagcgccactatgagccggtagta gctcgtcgggatggcgcggccgtctggtttttcagtttcatggcccgcacccccagggccggtgcgttgcacgtaactggttccgagc agactgtttccggcgtgtcgggaccccggagagtcaatggcgtgatccttaaccgctggctgtacttaatgtagcgctggttagggcga ccgtctggggcgttcggaggtcacccccacagcctcgtgggcacttcaggggattttccgccgtgctcggcccccagccacgtcctc gccgaggagttcgttgaggcgccctcctcgccggccccccgaccgggttgccggtttatatattcccccccaacgggggaagacccc cacggagggccgaaacggccatcgccacaccacgccagtcgaacagcatgttccagaccaccaggtgcggcccgtggccgagca gccttggagtctgccctacacgcacaaaccttcccacaccaccccccaactgaccccgtccccctcgtagtaccctccgggggtccac tctttgtggacggcggcccgacccagaccgacgaacgcggacgcacaggccatcaacacgatcttgattggcgacgaggtgcgcgt gcgtcgcgtgccgcctcggcccgtcaagcttggccgtgccccccctcggtcgcaggcgtcccgtctcgctaacggactggctgcgg cccgccctctgaccaggcctctgtggttagggcggtaaggagtgcgcgcccagggggtgcgtcacagattcgcgggccgcaccac caacagcggccctcgaacacgggcggggccacctggagctaaaaaatcagaggccacaacccgcgcacgaggtcggcgttggtc cagaacggcgtcggcactctggggcgcgctcacgttcggggatcgctggagcctggtggtgtcccacggtgcacgcgcggtgttgg gggtcgctcacgttgttggcatggccgcggccccagacagaagggcaggcgcagctggggcccgcaagcgggaaagaggggga cggttttggggttggggctggggtcccggtgccgaccaaccattacctctcggatgggggcgcgcctggtgacaccgcgcccctag gccgtgcacacgaactgggggtagctcaggagggcgtgtcgtcgagcgacccggggttgacccagcggctgatctccccttccctc ccccctgtgcgtgtaactgcaggacgagcccgaatgtggtttagctgaggcgcccgcgcagccgtcgggcgcgtcttggtacgtgcg ggccgcgcagtatgatcaggacccggtcaccttccggccccggcgccccggagtacacgataaacttggcgtgagcggcgtagcg cgtgaggcccaagaaggcgggggccgcggtagcgcacgtgaggcggagcagcgcgttggtctcggtgcagggatttaccgcgga gttcctcactgcctcgtacctgcggctcgtagccgccgaagggaccaatgacgagggcgagtcgacgcagacgcatagagccctga gtcacaggaaacagaaacgcgtcgcccctctccagctcttggaggattctcgactccacgcgcgttttcctccgcagttccaggcgact gcgtgctctccggtgacgttcatcgtcggcctggcactccggccgcaggcaggggcgggcggttggaccacgcggagcaagctgc gccagccgagttctcgtaggagagcgcggtgctgtaccggagtgcagtggtggcgttgtgaaacaggtaggccggtacacacccgt tgtcggcccgccttccgtggcttttttcgagctgacccagcgtccgtggcctagggcgaactcgatggaggcttgcgtggcgatgtgg cactgactcaggatatcctgtctgctctcccgccggccgcagcaggcatttggcacacccagtgcgttccggcgcgggacccgaggt cgccctcgatcgccctggcgtcctccagttccgcctttgcagagccaaggaagtggccctccgcgatcggtaacgcgaccaagcgg gggcgctggggtttggggtccgagggcgtgacgtcccagctgggggcggcgcgctcagtaacggccgtgggcgcgaagactcat cgcgcggaacgagctgacgccggtccgggtttccgctttatcctctcgggcatcgtttcccgccgcgaacccgacccgcgcctcgcc cgcccaggcggcgccggttcggaccaccagctcctaagcgccccgtcgatccaggaccaacgccgctaccgatgttccagaacaa acgctgctccgctaaaacctggggtcacgcgggcagtaaaacacgtccgcgggtcgttaacatcagcgaagcggcggtttagggtc agcagcgacgaccaaaccgactggggatcaccgacgctccttctcgtttccgacgtcctgcactgccattcgctcctatcacacgagc cgggccagcgcggcgggcgcggcgaaactcggccgtggtacctgtcggcggttgcgcggggtcgactcgtggcccgggccttga gcagggcgtcggccacccacacagcatgggcctggccgaccagacccgccttccagctggtcgtcagacgcgagaacttctcccg ggcggggtctgaccatacatcgaccgggacaccatttggtcccccctctccggcgattggagggaagcgcacggactcgggggttg atacgggacttcgaaactacccccgcccccgcgtattgcccttggtgtcggggggggcctgtggggagagcgggtggtccgtgcct cgcgctaccgctcctgttggtataggtcgtgcctggcgggcggccgccgcttgatcatgtagaaatcccgcaggagccccacgcaag gtgccccagctgcctgtgccgcggcctcacgggcgtccggcccgcgccagtgccgacgaacgtcccttcaaccactaactggccgc cgggcatccccgcggaacactgtagttgtttatgtggcgcgctgggcagggggggggcgcggggcggagctcggcaggcgccgt cttctggtaagcccttttattgatctggcgtctggagcggcatctcccggaaggctctccaaggcgaccaatcaggttggcaggcgggc ggaggacccggcctccagcgggtctgcgtggcgggcggcgaaagctgggggcgaagagatccgtgtcctgaggtggtcggcgtc ggtgtacagcagccgcccctcgggcgatttgccgtccgttccctgtcttctcccgccctccgcgtgcgtggtcggcgcctgcaggcga cgtcgcggcgagacggcaatagttattcggagtcgtcaccacgccgccgccctccttcggggtgccgctgtcgggacaggccacgt acgccagcgaggcgaccacccggctcgcgcgcgtcgacgtcgactccccgcctccgggagcctgtgggtccggcaggcgggtga actccacggccgagggaatggacgccttgctgggttgcggaggtggcgccggatgggacgctccacgactgctgcccgcaccgtc ccccgacgacaggggacggcctcctgaaccgcagacgagcttctcaactgggtcagccaggcgacgaggggtcgctggcgcagg tgagagcccgtccggactgaaggtagattgccaagtttggtctgtagcaaatgaatgacggttcttggtcgtaatccaggataaattcga ggccacacgcgtcagggacttttggagtcgccctgcgattttcgagcgccgcgtttgttgggaacttgggtgtgccgatacagccgtcg ggggccccaaacgggtctccccgcccccgccttgtttgaggcaggaccacgagcacaaacgtgcgagatcgagtggcgtctaacc cgactgcgcgctgcggtcgtccctctacgttgcggggtagcgtccgggcgcttggcagcgcccggcgggtccgctgtcctcgcggt ccgcctggcgtcgcgggacagggcctgcttccggccccagcgcagccacccaaagtgccccgaccccgagagtgggggggcct gggtccgcgtcctgcagggcgccgaactgacagccaccgggccgtgtcacgatcaccgcgggagcggtgcggcctgggttggcg tcgggtccttcgcgtttaagcttggtctcccatcagcagtttccaatcccggccccggggaatcagaagcggagcgggctggggacg gggacggcgtacgagaagggccagggcgaggtgcggcgcgctttagcgaccgacgcggcaccaacaacgacaccgagccggc ggagagccgccgccctcaccaacctgttcttacgtcgccggtaggggcagacgccccctgctccccggaccccggcgccggaacc gagccccgcgggggcgtggccaagccccccgcccctgggcacgcgcacggcgatcagagggtcagtggtgtttcctcatggccc cttatcgggggttcccaccaaacctgcggccaacacaacggtggatgcctctctcaagatggg

[0167] SEQ.5: 9kb Staffer

[0168] GC content: 67% ggtcgggcttggatttaatactaacgcgtaaatagtgagctttcacgcgcctccgcctgggagcgcagtctcggtggggatggcctctg tccggcgccgtcgccaggccggtcacgacgccctttttagaatgcaaggaccgtttactgagccttcgctcccgtctccacatagtgat aaaggcacacagcagacgaccgcgtaaggccgtggacgtagcgtttggccaagacgtcccctgggaccgcgccggagacggcg gcggacggccctgcctccggtaccgtatctccgcggacagtcggagtcgtagagttggctgactactacctcgccccagtcgacgaa ccggtacccctcgttcccgcaggccagacgcgcgctacgtcgccctactctccctccataacaccaataactctcagtaacaggcgcg tccggcttctgtgcccgatctctcgccgcccggggcccctctccagtgtcccgacattatccacggcccccgctcgtttgccccgctgc tcggtcgttatgtcggcccccacatcgggggtgatcattgccgcgcccgggggggaaaagcgagcagccggcgcccagatcgaaa tcgtttttgccctcgcagacctttgggtctcgcacctgaggggtgccccaaggtcgatcgccctcctctgaaaatgaaccagaacagca gcggggagtaacccacgtggccccgaccaccacaatcgccaggctgcggaagtcgggcccacgagccgccctccacggccccc gcccgccccctctcggcgatgtgggttgctcgtggacgcgtgcctggacc aacccc aactcc agcccc agtgtccccttttcgcggc a aagactggttgtccgtggccgaggacgtttcgttgcggcgaggtggacgttacgaacacgtcgatatatattaaaggtgcaggatgtgt tttcgtgggcctcttccagtataagggttggtcgcctgcggtcctgacggccaagcgtccaaccgtttattcgccccagattatcttcccg ctacgaccgcgcggcggagcaccgcctggttatacccgtgacgcccccgagttggtaccgtatgcgtgcgaattggtcggggccaa agcgggggagcggttttgaggtccagcgaattgagcgtgaagtttaccccggtagacaaacagggccgaattttaacacagaagcgc caccagacggtgaccgtatccacggccagtacgagcttcttcggggtgaatgttggcggcgatgtcgcggacatcccccaaagcga gctccgaccacgcttggggcgccaaacacgttatacagcgtgttcgaattcgcaggcccagcgcacagtaacgtggaccaccgttgg cctcgtcaaagcgccgggcgggtacgccacctccagggcgaccgggccaacccaatccggtctatgagcagcagcagctcgtcgg ctgacgcggccgttgcgcctttcagtttacaaccacccgcacctacgaccggtgcgtttagcgtgcccggtgcgcggggagactgttt ccagggggtcgggacgccagcgagtcaagctcagcatccttaacgatagacgtacttaactcgcttctggttaaagccgcgtctgggc agcccggaggtcacctcgcgggcctcgtgcagtcgtacaggggaagcgcctcgtgctcgcgccatcgccacgtcgatccaaggag ttctcgcgcccgccctcccccaatcccccccgagggccgagccggtttcgacgcacccccccaggactggaagaccccctggcag gccgaaaacagcaggcgccactgccctggtcgaacaccgaggcccagaccgccgctgcggcccgtatccgatacagccttgcacc caacacccccaaaccttccccgctgaagcccccaggctttgccgtccccctgaccgaccctccggtatcaaactcttttacggggcgc ggccccgcaggcgtgcctgggcgatttccgacgaagcaccagaaagcacaaaccgcgccctccccgtctgacgatctgcaccccc gacgagggttggccgtcgcgtggcccgagcggcccgtgcacacctggccgtggataaggcggtcgcagtggtccgtctcgctccg ccgtctggctgccgtgggcctctgaccagttggctgtggttaactgcggtttaaggacggggtcagggggtgctgtgagattcgcgct cgaccaccaccaagcttggccctcgaacttcgtgccgggccaccgaactgacaaaaaattgacgcccacaacccggtccggaggtc gtgaggagccagaacgctccggtcacgttccgggaccgctggagcagcgccggtgtcccataatacagcgcggttcgccagctcg ctcagcgagcgctggccgtctggtccagacaggcggcagcgcgcagctcgcccgcaagcgccaggggggacgggggttggggt tggggctgcgccattaccacgcgggtgggggcgggagcaatacaccgccgggacgaggccgtggccaggaactgggggcccgc gaggagggcaggacccagagcgaccgctgccgacccagtcctcttctccccttgcacctcccccttagcagccgctgcaggtgctc gcgaattgggggtggcggaggcgcgagggccgtcgggcaggcgtggtacgtgcgccgcggcgcagtattgagacggacccggg gacggacggccccgtcggcgggagagtactgggcggcttggcggatgggcaggtagcgcgtgcgcacgcggtagccaggccca gaggcgggggctgggttggtggccacgggatttactgttctcgttcctccggcacgtacctgcaggccaagccgccaggatgacaat gacttaaggcgtcgacgccagagggtagagccggtgtagtggcagtacggtgccgcgcgggtgttaacggtcagcgagtatgcagt ttaggggaggtggcggacgacgcagcgcgttggggaggaaaccatctccttgcggcgggtgacgtcctccgcacacattcgatgca ctggaccacgtgaccgcggagcggcgtcccgcggaaactccattccacagtacctgcgggccgcgcgggtcggcgccgtggccg cgcctccagcagggggaaaacccactgcctgctgcgcctgctcgaacgcccccgccgccggagtcgtcaggatgtcccagtgggt ggaccctgttcgaccctatatagtttggtggaggaaccggcgatctagggcggagcgcccattgggtggggttcaaagcgcttcgac ctgacgcccgcccccgactacgcgaccctggcgccgcggggggcgacggggaacagcggaatcgcgcctcacctcccaacgcc tcccctgttcacattgccccgccgccgcggagccgtagagctcgcccccgagcccactcccccgtatctctttgtgctgtgccgatcgc gcaaggcggcgctggaagactgccagtgcggggcccgtccctcgcggctcaactggctcgctgtcgtcatccttcctgtgtgtagttc ccgaccacggcgcgcctctgcgtggggggccgcggagctcgcctccgattcgtccgcctcacgccccctacgcggatcgggcgat acctcctctcatacctccatctcgtcgccgcggatcagggagtgggcggaggcctcctcggcctccatcggcgtcggcgtggcggga ccaaaaagcttccccccgggagatcgtcgtcgtcaggtgcgtccggggtgtacacgcgcgaaatccctcgccagctcgccgtccttct tccctcttctcccgcgcctcgcgtggcccgccagcgcctggagccagctgtcgcggtgcaagaacttctcgtgcgggggggcttgcg gcgacctcggcgcaggccctgggagcgcctcgaggcggactggcgccagccgcggtttggtatagatcgacgggagtcagcgttg ggacgccacggcgagctccgcgctggtgcggtacagggctgggtgcaaacacaggtcgcgcgcaccagaccgtggtcgtatcca cgggagggacgattgttaagccagctttttgtacaacgggtgaatgagctggtgtcggccgacgtaaatcccccaaaacacgagctcc gacaacgcgggcgccctccgtcgggttatacaggtcgcccgaattcgccaatgaagcgcacagcggctgggaccaccgcgctggt cgtcaaagtcgccggggcgggtaacggtactccagggccgcgtcgccaacccagcgccactatgagccggtagtagctcgtcggg atggcgcggccgtctggtttttcagtttcatggcccgcacccccagggccggtgcgttgcacgtaactggttccgagcagactgtttcc ggcgtgtcgggaccccggagagtcaatggcgtgatccttaaccgctggctgtacttaatgtagcgctggttagggcgaccgtctgggg cgttcggaggtcacccccacagcctcgtgggcacttcaggggattttccgccgtgctcggcccccagccacgtcctcgccgaggagt tcgttgaggcgccctcctcgccggccccccgaccgggttgccggtttatatattcccccccaacgggggaagacccccacggaggg ccgaaacggccatcgccacaccacgccagtcgaacagcatgttccagaccaccaggtgcggcccgtggccgagcagccttggagt ctgccctacacgcacaaaccttcccacaccaccccccaactgaccccgtccccctcgtagtaccctccgggggtccactctttgtgga cggcggcccgacccagaccgacgaacgcggacgcacaggccatcaacacgatcttgattggcgacgaggtgcgcgtgcgtcgcg tgccgcctcggcccgtcaagcttggccgtgccccccctcggtcgcaggcgtcccgtctcgctaacggactggctgcggcccgccctc tgaccaggcctctgtggttagggcggtaaggagtgcgcgcccagggggtgcgtcacagattcgcgggccgcaccaccaacagcgg ccctcgaacacgggcggggccacctggagctaaaaaatcagaggccacaacccgcgcacgaggtcggcgttggtccagaacggc gtcggcactctggggcgcgctcacgttcggggatcgctggagcctggtggtgtcccacggtgcacgcgcggtgttgggggtcgctc acgttgttggcatggccgcggccccagacagaagggcaggcgcagctggggcccgcaagcgggaaagagggggacggttttgg ggttggggctggggtcccggtgccgaccaaccattacctctcggatgggggcgcgcctggtgacaccgcgcccctaggccgtgca cacgaactgggggtagctcaggagggcgtgtcgtcgagcgacccggggttgacccagcggctgatctccccttccctcccccctgtg cgtgtaactgcaggacgagcccgaatgtggtttagctgaggcgcccgcgcagccgtcgggcgcgtcttggtacgtgcgggccgcgc agtatgatcaggacccggtcaccttccggccccggcgccccggagtacacgataaacttggcgtgagcggcgtagcgcgtgaggc ccaagaaggcgggggccgcggtagcgcacgtgaggcggagcagcgcgttggtctcggtgcagggatttaccgcggagttcctcac tgcctcgtacctgcggctcgtagccgccgaagggaccaatgacgagggcgagtcgacgcagacgcatagagccctgagtcacagg aaacagaaacgcgtcgcccctctccagctcttggaggattctcgactccacgcgcgttttcctccgcagttccaggcgactgcgtgctc tccggtgacgttcatcgtcggcctggcactccggccgcaggcaggggcgggcggttggaccacgcggagcaagctgcgccagcc gagttctcgtaggagagcgcggtgctgtaccggagtgcagtggtggcgttgtgaaacaggtaggccggtacacacccgttgtcggcc cgccttccgtggcttttttcgagctgacccagcgtccgtggcctagggcgaactcgatggaggcttgcgtggcgatgtggcactgactc aggatatcctgtctgctctcccgccggccgcagcaggcatttggcacacccagtgcgttccggcgcgggacccgaggtcgccctcg atcgccctggcgtcctccagttccgcctttgcagagccaaggaagtggccctccgcgatcggtaacgcgaccaagcgggggcgctg gggtttggggtccgagggcgtgacgtcccagctgggggcggcgcgctcagtaacggccgtgggcgcgaagactcatcgcgcgga acgagctgacgccggtccgggtttccgctttatcctctcgggcatcgtttcccgccgcgaacccgacccgcgcctcgcccgcccagg cggcgccggttcggaccaccagctcctaagcgccccgtcgatccaggaccaacgccgctaccgatgttccagaacaaacgctgctc cgctaaaacctggggtcacgcgggcagtaaaacacgtccgcgggtcgttaacatcagcgaagcggcggtttagggtcagcagcga cgaccaaaccgactggggatcaccgacgctccttctcgtttccgacgtcctgcactgccattcgctcctatcacacgagccgggccag cgcggcgggcgcggcgaaactcggccgtggtacctgtcggcggttgcgcggggtcgactcgtggcccgggccttgagcagggcg tcggccacccacacagcatgggcctggccgaccagacccgccttccagctggtcgtcagacgcgagaacttctcccgggcggggt ctgaccatacatcgaccgggacaccatttggtcccccctctccggcgattggagggaagcgcacggactcgggggttgatacggga cttcgaaactacccccgcccccgcgtattgcccttggtgtcggggggggcctgtggggagagcgggtggtccgtgcctcgcgctacc gctcctgttggtataggtcgtgcctggcgggcggccgccgcttgatcatgtagaaatcccgcaggagccccacgcaaggtgccccag ctgcctgtgccgcggcctcacgggcgtccggcccgcgccagtgccgacgaacgtcccttcaaccactaactggccgccgggcatcc ccgcggaacactgtagttgtttatgtggcgcgctgggcagggggggggcgcggggcggagctcggcaggcgccgtcttctggtaa gcccttttattgatctggcgtctggagcggcatctcccggaaggctctccaaggcgaccaatcaggttggcaggcgggcggaggacc cggcctccagcgggtctgcgtggcgggcggcgaaagctgggggcgaagagatccgtgtcctgaggtggtcggcgtcggtgtacag cagccgcccctcgggcgatttgccgtccgttccctgtcttctcccgccctccgcgtgcgtggtcggcgcctgcaggcgacgtcgcgg cgagacggcaatagttattcggagtcgtcaccacgccgccgccctccttcggggtgccgctgtcgggacaggccacgtacgccagc gaggcgaccacccggctcgcgcgcgtcgacgtcgactccccgcctccgggagcctgtgggtccggcaggcgggtgaactccacg gccgagggaatggacgccttgctgggttgcggaggtggcgccggatgggacgctccacgactgctgcccgcaccgtcccccgacg acaggggacggcctcctgaaccgcagacgagcttctcaactgggtcagccaggcgacgaggggtcgctggcgcaggtgagagcc cgtccggactgaaggtagattgccaagtttggtctgtagcaaatgaatgacggttcttggtcgtaatccaggataaattcgaggccacac gcgtcagggacttttggagtcgccctgcgattttcgagcgccgcgtttgttgggaacttgggtgtgccgatacagccgtcgggggccc caaacgggtctccccgcccccgccttgtttgaggcaggaccacgagcacaaacgtgcgagatcgagtggcgtctaacccgactgcg cgctgcggtcgtccctctacgttgcggggtagcgtccgggcgcttggcagcgcccggcgggtccgctgtcctcgcggtccgcctgg cgtcgcgggacagggcctgcttccggccccagcgcagccacccaaagtgccccgaccccgagagtgggggggcctgggtccgc gtcctgcagggcgccgaactgacagccaccgggccgtgtcacgatcaccgcgggagcggtgcggcctgggttggcgtcgggtcct tcgcgtttaagcttggtctcccatcagcagtttccaatcccggccccggggaatcagaagcggagcgggctggggacggggacggc gtacgagaagggccagggcgaggtgcggcgcgctttagcgaccgacgcggcaccaacaacgacaccgagccggcggagagcc gccgccctcaccaacctgttcttacgtcgccggtaggggcagacgccccctgctccccggaccccggcgccggaaccgagccccg cgggggcgtggccaagccccccgcccctgggcacgcgcacggcgatcagagggtcagtggtgtttcctcatggccccttatcggg ggttcccaccaaacctgcggccaacacaacggtggatgcctctctcaagatggg

[0169] SEQ.6: 6kb Staffer

[0170] GC content: 67% ggtcgggcttggatttaatactaacgcgtaaatagtgagctttcacgcgcctccgcctgggagcgcagtctcggtggggatggcctctg tccggcgccgtcgccaggccggtcacgacgccctttttagaatgcaaggaccgtttactgagccttcgctcccgtctccacatagtgat aaaggcacacagcagacgaccgcgtaaggccgtggacgtagcgtttggccaagacgtcccctgggaccgcgccggagacggcg gcggacggccctgcctccggtaccgtatctccgcggacagtcggagtcgtagagttggctgactactacctcgccccagtcgacgaa ccggtacccctcgttcccgcaggccagacgcgcgctacgtcgccctactctccctccataacaccaataactctcagtaacaggcgcg tccggcttctgtgcccgatctctcgccgcccggggcccctctccagtgtcccgacattatccacggcccccgctcgtttgccccgctgc tcggtcgttatgtcggcccccacatcgggggtgatcattgccgcgcccgggggggaaaagcgagcagccggcgcccagatcgaaa tcgtttttgccctcgcagacctttgggtctcgcacctgaggggtgccccaaggtcgatcgccctcctctgaaaatgaaccagaacagca gcggggagtaacccacgtggccccgaccaccacaatcgccaggctgcggaagtcgggcccacgagccgccctccacggccccc gcccgccccctctcggcgatgtgggttgctcgtggacgcgtgcctggacc aacccc aactcc agcccc agtgtccccttttcgcggc a aagactggttgtccgtggccgaggacgtttcgttgcggcgaggtggacgttacgaacacgtcgatatatattaaaggtgcaggatgtgt tttcgtgggcctcttccagtataagggttggtcgcctgcggtcctgacggccaagcgtccaaccgtttattcgccccagattatcttcccg ctacgaccgcgcggcggagcaccgcctggttatacccgtgacgcccccgagttggtaccgtatgcgtgcgaattggtcggggccaa agcgggggagcggttttgaggtccagcgaattgagcgtgaagtttaccccggtatgtcggccgacgtaaatcccccaaaacacgagc tccgacaacgcgggcgccctccgtcgggttatacaggtcgcccgaattcgccaatgaagcgcacagcggctgggaccaccgcgct ggtcgtcaaagtcgccggggcgggtaacggtactccagggccgcgtcgccaacccagcgccactatgagccggtagtagctcgtc gggatggcgcggccgtctggtttttcagtttcatggcccgcacccccagggccggtgcgttgcacgtaactggttccgagcagactgtt tccggcgtgtcgggaccccggagagtcaatggcgtgatccttaaccgctggctgtacttaatgtagcgctggttagggcgaccgtctg gggcgttcggaggtcacccccacagcctcgtgggcacttcaggggattttccgccgtgctcggcccccagccacgtcctcgccgag gagttcgttgaggcgccctcctcgccggccccccgaccgggttgccggtttatatattcccccccaacgggggaagacccccacgga gggccgaaacggccatcgccacaccacgccagtcgaacagcatgttccagaccaccaggtgcggcccgtggccgagcagccttg gagtctgccctacacgcacaaaccttcccacaccaccccccaactgaccccgtccccctcgtagtaccctccgggggtccactctttgt ggacggcggcccgacccagaccgacgaacgcggacgcacaggccatcaacacgatcttgattggcgacgaggtgcgcgtgcgtc gcgtgccgcctcggcccgtcaagcttggccgtgccccccctcggtcgcaggcgtcccgtctcgctaacggactggctgcggcccgc cctctgaccaggcctctgtggttagggcggtaaggagtgcgcgcccagggggtgcgtcacagattcgcgggccgcaccaccaaca gcggccctcgaacacgggcggggccacctggagctaaaaaatcagaggccacaacccgcgcacgaggtcggcgttggtccagaa cggcgtcggcactctggggcgcgctcacgttcggggatcgctggagcctggtggtgtcccacggtgcacgcgcggtgttgggggtc gctcacgttgttggcatggccgcggccccagacagaagggcaggcgcagctggggcccgcaagcgggaaagagggggacggtt ttggggttggggctggggtcccggtgccgaccaaccattacctctcggatgggggcgcgcctggtgacaccgcgcccctaggccgt gcacacgaactgggggtagctcaggagggcgtgtcgtcgagcgacccggggttgacccagcggctgatctccccttccctcccccc tgtgcgtgtaactgcaggacgagcccgaatgtggtttagctgaggcgcccgcgcagccgtcgggcgcgtcttggtacgtgcgggcc gcgcagtatgatcaggacccggtcaccttccggccccggcgccccggagtacacgataaacttggcgtgagcggcgtagcgcgtg aggcccaagaaggcgggggccgcggtagcgcacgtgaggcggagcagcgcgttggtctcggtgcagggatttaccgcggagttc ctcactgcctcgtacctgcggctcgtagccgccgaagggaccaatgacgagggcgagtcgacgcagacgcatagagccctgagtc acaggaaacagaaacgcgtcgcccctctccagctcttggaggattctcgactccacgcgcgttttcctccgcagttccaggcgactgc gtgctctccggtgacgttcatcgtcggcctggcactccggccgcaggcaggggcgggcggttggaccacgcggagcaagctgcgc cagccgagttctcgtaggagagcgcggtgctgtaccggagtgcagtggtggcgttgtgaaacaggtaggccggtacacacccgttgt cggcccgccttccgtggcttttttcgagctgacccagcgtccgtggcctagggcgaactcgatggaggcttgcgtggcgatgtggcac tgactcaggatatcctgtctgctctcccgccggccgcagcaggcatttggcacacccagtgcgttccggcgcgggacccgaggtcgc cctcgatcgccctggcgtcctccagttccgcctttgcagagccaaggaagtggccctccgcgatcggtaacgcgaccaagcggggg cgctggggtttggggtccgagggcgtgacgtcccagctgggggcggcgcgctcagtaacggccgtgggcgcgaagactcatcgc gcggaacgagctgacgccggtccgggtttccgctttatcctctcgggcatcgtttcccgccgcgaacccgacccgcgcctcgcccgc ccaggcggcgccggttcggaccaccagctcctaagcgccccgtcgatccaggaccaacgccgctaccgatgttccagaacaaacg ctgctccgctaaaacctggggtcacgcgggcagtaaaacacgtccgcgggtcgttaacatcagcgaagcggcggtttagggtcagc agcgacgaccaaaccgactggggatcaccgacgctccttctcgtttccgacgtcctgcactgccattcgctcctatcacacgagccgg gccagcgcggcgggcgcggcgaaactcggccgtggtacctgtcggcggttgcgcggggtcgactcgtggcccgggccttgagca gggcgtcggccacccacacagcatgggcctggccgaccagacccgccttccagctggtcgtcagacgcgagaacttctcccgggc ggggtctgaccatacatcgaccgggacaccatttggtcccccctctccggcgattggagggaagcgcacggactcgggggttgatac gggacttcgaaactacccccgcccccgcgtattgcccttggtgtcggggggggcctgtggggagagcgggtggtccgtgcctcgcg ctaccgctcctgttggtataggtcgtgcctggcgggcggccgccgcttgatcatgtagaaatcccgcaggagccccacgcaaggtgc cccagctgcctgtgccgcggcctcacgggcgtccggcccgcgccagtgccgacgaacgtcccttcaaccactaactggccgccgg gcatccccgcggaacactgtagttgtttatgtggcgcgctgggcagggggggggcgcggggcggagctcggcaggcgccgtcttc tggtaagcccttttattgatctggcgtctggagcggcatctcccggaaggctctccaaggcgaccaatcaggttggcaggcgggcgga ggacccggcctccagcgggtctgcgtggcgggcggcgaaagctgggggcgaagagatccgtgtcctgaggtggtcggcgtcggt gtacagcagccgcccctcgggcgatttgccgtccgttccctgtcttctcccgccctccgcgtgcgtggtcggcgcctgcaggcgacgt cgcggcgagacggcaatagttattcggagtcgtcaccacgccgccgccctccttcggggtgccgctgtcgggacaggccacgtacg ccagcgaggcgaccacccggctcgcgcgcgtcgacgtcgactccccgcctccgggagcctgtgggtccggcaggcgggtgaact ccacggccgagggaatggacgccttgctgggttgcggaggtggcgccggatgggacgctccacgactgctgcccgcaccgtcccc cgacgacaggggacggcctcctgaaccgcagacgagcttctcaactgggtcagccaggcgacgaggggtcgctggcgcaggtga gagcccgtccggactgaaggtagattgccaagtttggtctgtagcaaatgaatgacggttcttggtcgtaatccaggataaattcgagg ccacacgcgtcagggacttttggagtcgccctgcgattttcgagcgccgcgtttgttgggaacttgggtgtgccgatacagccgtcggg ggccccaaacgggtctccccgcccccgccttgtttgaggcaggaccacgagcacaaacgtgcgagatcgagtggcgtctaacccga ctgcgcgctgcggtcgtccctctacgttgcggggtagcgtccgggcgcttggcagcgcccggcgggtccgctgtcctcgcggtccg cctggcgtcgcgggacagggcctgcttccggccccagcgcagccacccaaagtgccccgaccccgagagtgggggggcctgggt ccgcgtcctgcagggcgccgaactgacagccaccgggccgtgtcacgatcaccgcgggagcggtgcggcctgggttggcgtcgg gtccttcgcgtttaagcttggtctcccatcagcagtttccaatcccggccccggggaatcagaagcggagcgggctggggacgggga cggcgtacgagaagggccagggcgaggtgcggcgcgctttagcgaccgacgcggcaccaacaacgacaccgagccggcggag agccgccgccctcaccaacctgttcttacgtcgccggtaggggcagacgccccctgctccccggaccccggcgccggaaccgagc cccgcgggggcgtggccaagccccccgcccctgggcacgcgcacggcgatcagagggtcagtggtgtttcctcatggccccttatc gggggttcccaccaaacctgcggccaacacaacggtggatgcctctctcaagatggg

[0171] SEQ.7: 3kb Staffer

[0172] GC content: 67% ggtcgggcttggatttaatactaacgcgtaaatagtgagctttcacgcgcctccgcctgggagcgcagtctcggtggggatggcctctg tccggcgccgtcgccaggccggtcacgacgccctttttagaatgcaaggaccgtttactgagccttcgctcccgtctccacatagtgat aaaggcacacagcagacgaccgcgtaaggccgtggacgtagcgtttggccaagacgtcccctgggaccgcgccggagacggcg gcggacggccctgcctccggtaccgtatctccgcggacagtcggagtcgtagagttggctgactactacctcgccccagtcgacgaa ccggtacccctcgttcccgcaggccagacgcgcgctacgtcgccctactctccctccataacaccaataactctcagtaacaggcgcg tccggcttctgtgcccgatctctcgccgcccggggcccctctccagtgtcccgacattatccacggcccccgctcgtttgccccgctgc tcggtcgttatgtcggcccccacatcgggggtgatcattgccgcgcccgggggggaaaagcgagcagccggcgcccagatcgaaa tcgtttttgccctcgcagacctttgggtctcgcacctgaggggtgccccaaggtcgatcgccctcctctgaaaatgaaccagaacagca gcggggagtaacccacgtggccccgaccaccacaatcgccaggctgcggaagtcgggcccacgagccgccctccacggccccc gcccgccccctctcggcgatgtgggttgctcgtggacgcgtgcctggacc aacccc aactcc agcccc agtgtccccttttcgcggc a aagactggttgtccgtggccgaggacgtttcgttgcggcgaggtggacgttacgaacacgtcgatatatattaaaggtgcaggatgtgt tttcgtgggcctcttccagtataagggttggtcgcctgcggtcctgacggccaagcgtccaaccgtttattcgccccagattatcttcccg ctacgaccgcgcggcggagcaccgcctggttatacccgtgacgcccccgagttggtaccgtatgcgtgcgaattggtcggggccaa agcgggggagcggttttgaggtccagcgaattgagcgtgaagtttaccccggtaggggggcctgtggggagagcgggtggtccgt gcctcgcgctaccgctcctgttggtataggtcgtgcctggcgggcggccgccgcttgatcatgtagaaatcccgcaggagccccacg caaggtgccccagctgcctgtgccgcggcctcacgggcgtccggcccgcgccagtgccgacgaacgtcccttcaaccactaactgg ccgccgggcatccccgcggaacactgtagttgtttatgtggcgcgctgggcagggggggggcgcggggcggagctcggcaggcg ccgtcttctggtaagcccttttattgatctggcgtctggagcggcatctcccggaaggctctccaaggcgaccaatcaggttggcaggc gggcggaggacccggcctccagcgggtctgcgtggcgggcggcgaaagctgggggcgaagagatccgtgtcctgaggtggtcg gcgtcggtgtacagcagccgcccctcgggcgatttgccgtccgttccctgtcttctcccgccctccgcgtgcgtggtcggcgcctgca ggcgacgtcgcggcgagacggcaatagttattcggagtcgtcaccacgccgccgccctccttcggggtgccgctgtcgggacaggc cacgtacgccagcgaggcgaccacccggctcgcgcgcgtcgacgtcgactccccgcctccgggagcctgtgggtccggcaggcg ggtgaactccacggccgagggaatggacgccttgctgggttgcggaggtggcgccggatgggacgctccacgactgctgcccgca ccgtcccccgacgacaggggacggcctcctgaaccgcagacgagcttctcaactgggtcagccaggcgacgaggggtcgctggc gcaggtgagagcccgtccggactgaaggtagattgccaagtttggtctgtagcaaatgaatgacggttcttggtcgtaatccaggataa attcgaggccacacgcgtcagggacttttggagtcgccctgcgattttcgagcgccgcgtttgttgggaacttgggtgtgccgatacag ccgtcgggggccccaaacgggtctccccgcccccgccttgtttgaggcaggaccacgagcacaaacgtgcgagatcgagtggcgt ctaacccgactgcgcgctgcggtcgtccctctacgttgcggggtagcgtccgggcgcttggcagcgcccggcgggtccgctgtcctc gcggtccgcctggcgtcgcgggacagggcctgcttccggccccagcgcagccacccaaagtgccccgaccccgagagtggggg ggcctgggtccgcgtcctgcagggcgccgaactgacagccaccgggccgtgtcacgatcaccgcgggagcggtgcggcctgggt tggcgtcgggtccttcgcgtttaagcttggtctcccatcagcagtttccaatcccggccccggggaatcagaagcggagcgggctggg gacggggacggcgtacgagaagggccagggcgaggtgcggcgcgctttagcgaccgacgcggcaccaacaacgacaccgagc cggcggagagccgccgccctcaccaacctgttcttacgtcgccggtaggggcagacgccccctgctccccggaccccggcgccgg aaccgagccccgcgggggcgtggccaagccccccgcccctgggcacgcgcacggcgatcagagggtcagtggtgtttcctcatg gccccttatcgggggttcccaccaaacctgcggccaacacaacggtggatgcctctctcaagatggg

[0173] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS:

1. A modified HSV-1 vector comprising one or more nucleic acid sequences of interest and one or more stuffers, wherein the size of the modified HSV-1 vector genome is about 142 kbp to about 160 kbp.

2. The modified HSV-1 vector according to claim 1, wherein the one or more nucleic acid sequences of interest comprises one or more transgenes of interest, one or more nucleic acids encoding a cell targeting protein, or combinations thereof.

3. The modified HSV-1 vector according to claim 2, wherein the one or more nucleic acid sequences of interest comprises one or more transgenes of interest.

4. The modified HSV-1 vector according to claim 3, wherein the one or more transgene of interest is operably linked to at least one sequence conferring long-term expression.

5. The modified HSV-1 vector according to any one of claims 1-4, wherein the size of the modified HSV-1 genome is about 143 kbp to about 158 kbp.

6. The modified HSV-1 vector according to claim 5, wherein the size of the modified HSV-1 genome is about 145 kbp to about 155 kbp.

7. The modified HSV-1 vector according to claim 6, wherein the size of the modified HSV-1 genome is about 152 kbp.

8. The modified HSV-1 vector according to any one of claims 1 to 7, wherein the one or more stuffers independently comprises non-coding DNA with GC content of 60-75%.

9. The modified HSV-1 vector according to claim 8, wherein the one or more stuffers are independently selected from a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 7.

10. The modified HSV-1 vector according to any one of claims 2 to 9, wherein the one or more transgenes of interest are independently part of an expression cassette.

11. The modified HSV-1 vector according to claim 10, wherein the expression cassette is operably linked to at least one sequence conferring long-term expression.

12. The modified HSV-1 vector according to any one of claims 4 to 11, wherein the at least one sequence conferring long-term expression is selected from LTE and / or DNA insulators from the HSV-1 genome.

13. A method for making a modified HSV-1 vector comprising: a) removing non-essential genes, essential genes, or combinations thereof from an HSV-1 genome to arrive at a HSV-1 pre-vector backbone genome comprising less than 130 kbp and greater than 75 kbp; b) inserting into the HSV-1 pre- vector backbone genome one or more nucleic acid sequences of interest; and c) inserting one or more stuffers into the HSV-1 pre- vector backbone genome, wherein the size of the modified HSV-1 vector genome after b) and c) is about 142 kbp to about 160 kbp.

14. The method according to claim 13, wherein the one or more nucleic acid sequences of interest comprises one or more transgenes of interest, one or more nucleic acids encoding a cell targeting protein, or combinations thereof.

15. The method according to claim 14, wherein the one or more nucleic acid sequences of interest comprises one or more transgenes of interest.

16. The modified HSV-1 vector according to claim 15, wherein the one or more transgene of interest is operably linked to at least one sequence conferring long-term expression.

17. The method according to any one of claims 13-16, wherein the size of the modified HSV-1 genome after b) and c) is about 143 kbp to about 158 kbp.

18. The method according to claim 17, wherein the size of the modified HSV-1 genome after b) and c) is about 147 kbp to about 155 kbp.

19. The method according to claim 18, wherein the size of the modified HSV-1 genome after b) and c) is about 152 kbp.

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