METHODS AND PRODUCTS FOR PRODUCING ENGINEERED MAMMALIAN CELL LINES WITH AMPLIFIED TRANSGENES
20190249199 ยท 2019-08-15
Assignee
Inventors
- Derek Jantz (Durham, NC)
- James Jefferson Smith (Morrisville, NC)
- Michael G. Nicholson (Chapel Hill, NC)
Cpc classification
C12N9/22
CHEMISTRY; METALLURGY
International classification
C12N15/90
CHEMISTRY; METALLURGY
C12N9/00
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
Abstract
Methods of inserting genes into defined locations in the chromosomal DNA of cultured mammalian cell lines which are subject to gene amplification are disclosed. In particular, sequences of interest (e.g., genes encoding biotherapeutic proteins) are inserted proximal to selectable genes in amplifiable loci, and the transformed cells are subjected to selection to induce co-amplification of the selectable gene and the sequence of interest. The invention also relates to meganucleases, vectors and engineered cell lines necessary for performing the methods, to cell lines resulting from the application of the methods, and use of the cell lines to produce protein products of interest.
Claims
1-19. (canceled)
20. A method for inserting an exogenous sequence into an amplifiable locus of a mammalian cell comprising: (a) providing a mammalian cell having an endogenous target site proximal to a selectable gene within the amplifiable locus, wherein the endogenous target site comprises: (i) a recognition sequence for an engineered meganuclease; (ii) a 5 flanking region 5 to the recognition sequence; and (iii) a 3 flanking region 3 to the recognition sequence; and (b) introducing a double-stranded break between the 5 and 3 flanking regions of the endogenous target site; (c) contacting the cell with a donor vector comprising from 5 to 3: (i) a donor 5 flanking region homologous to the 5 flanking region of the endogenous target site; (ii) an exogenous sequence; and (iii) a donor 3 flanking region homologous to the 3 flanking region of the endogenous target site; whereby the donor 5 flanking region, the exogenous sequence and the donor 3 flanking region are inserted between the 5 and 3 flanking regions of the endogenous target site by homologous recombination to provide a modified cell.
21. The method of claim 20, further comprising growing the modified cell in the presence of a compound that inhibits the function of the selectable gene to amplify the copy number of the selectable gene.
22. The method of claim 20, wherein the exogenous sequence comprises a gene of interest.
23. The method of claim 20, wherein the endogenous target site is downstream from the 3 regulatory region of the selectable gene.
24. The method of claim 23, wherein the endogenous target site is 0 to 100,000 base pairs downstream from the 3 regulatory region of the selectable gene.
25. The method of claim 20, wherein the endogenous target site is upstream from the 5 regulatory region of the selectable gene.
26. The method of claim 25, wherein the endogenous target site is 0 to 100,000 base pairs upstream from the 5 regulatory region of the selectable gene.
27. The method of claim 20, wherein the selectable gene is glutamine synthetase (GS) and the locus is methionine sulphoximine (MSX) amplifiable.
28. The method of claim 20, wherein the selectable gene is dihydrofolate reductase (DHFR) and the locus is Methotrexate (MTX) amplifiable.
29. The method of claim 20, wherein the selectable gene is selected from the group consisting of Dihydrofolate Reductase, Glutamine Synthetase, Hypoxanthine Phosphoribosyltransferase, Threonyl tRNA Synthetase, Na,K-ATPase, Asparagine Synthetase, Ornithine Decarboxylase, Inosine-5-monophosphate dehydrogenase, Adenosine Deaminase, Thymidylate Synthetase, Aspartate Transcarbamylase, Metallothionein, Adenylate Deaminase (1,2), UMP-Synthetase and Ribonucleotide Reductase.
30. The method of claim 29, wherein the selectable gene is amplifiable by selection with a selection agent selected from the group consisting of Methotrexate (MTX), Methionine sulphoximine (MSX), Aminopterin, hypoxanthine, thymidine, Borrelidin, Ouabain, Albizziin, Beta-aspartyl hydroxamate, alpha-difluoromethylornithine (DFMO), Mycophenolic Acid, Adenosine, Alanosine, 2deoxycoformycin, Fluorouracil, N-Phosphonacetyl-L-Aspartate (PALA), Cadmium, Adenine, Azaserine, Coformycin, 6-azauridine, pyrazofuran, hydroxyurea, motexafin gadolinium, fludarabine, cladribine, gemcitabine, tezacitabine and triapine.
31-54. (canceled)
55. A recombinant meganuclease comprising a polypeptide having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 9.
56. The recombinant meganuclease of claim 55, having the sequence of the meganuclease of SEQ ID NO: 9.
57. A recombinant meganuclease which recognizes and cleaves a recognition site having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7.
58. The recombinant meganuclease of claim 57, wherein the meganuclease recognizes and cleaves a recognition site of SEQ ID NO: 7.
59-70. (canceled)
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
1.1 Introduction
[0055] The present invention depends, in part, upon the development of mammalian cell lines in which exogenous actively transcribed transgenes have been inserted proximal to an endogenous amplifiable locus, and the discovery that (a) the insertion of such exogenous actively transcribed transgenes does not prevent or substantially inhibit amplification of the endogenous amplifiable locus, (b) the exogenous actively transcribed transgene can be co-amplified with the endogenous amplifiable locus, and (c) the resultant cell line, with an amplified region comprising multiple copies of the endogenous amplifiable locus and the exogenous actively transcribed transgene is stable for extended periods even in the absence of the selection regime which was employed to induce amplification. Thus, in one aspect, the invention provides a method for producing cell lines which can be used for biomanufacturing of a protein product of interest by specifically targeting the insertion of an exogenous gene capable of actively expressing the protein product of interest proximal to an endogenous amplifiable locus. In another aspect, the invention provides engineered cell lines that can be used to produce protein products of interest (e.g., therapeutic proteins such as monoclonal antibodies) at high levels.
1.2 References and Definitions
[0056] The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The entire disclosures of the issued U.S. patents, pending applications, published foreign applications, and scientific and technical references cited herein, including protein and nucleic acid database sequences, are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.
[0057] As used herein, the term meganuclease refers to naturally-occurring homing endonucleases (also referred to as Group I intron encoded endonucleases) or non-naturally-occurring (e.g., rationally designed or engineered) endonucleases based upon the amino acid sequence of a naturally-occurring homing endonuclease. Examples of naturally-occurring meganucleases include I-SceI, I-CreI, I-CeuI, I-DmoI, I-MsoI, I-AniI, etc. Rationally designed meganucleases are disclosed in, for example, WO 2007/047859 and WO 2009/059195, and can be engineered to have modified DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties relative to a naturally occurring meganuclease. A meganuclease may bind to double-stranded DNA as a homodimer (e.g., wild-type I-CreI), or it may bind to DNA as a heterodimer (e.g., engineered meganucleases disclosed in WO 2007/047859). An engineered meganuclease may also be a single-chain meganuclease in which a pair of DNA-binding domains derived from a natural meganuclease are joined into a single polypeptide using a peptide linker (e.g., single-chain meganucleases disclosed in WO 2009/059195).
[0058] As used herein, the term single-chain meganuclease refers to a polypeptide comprising a pair of meganuclease subunits joined by a linker. A single-chain meganuclease has the organization: N-terminal subunit-Linker-C-terminal subunit. The two meganuclease subunits will generally be non-identical in amino acid sequence and will recognize non-identical DNA sequences. Thus, single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. Methods of producing single-chain meganucleases are disclosed in WO 2009/059195.
[0059] As used herein, the term site specific endonuclease means a meganuclease, zinc-finger nuclease or TAL effector nuclease.
[0060] As used herein, with respect to a protein, the term recombinant means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the protein, and cells or organisms which express the protein. With respect to a nucleic acid, the term recombinant means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally-occurring protein, but produced by cloning and expression in a heterologous host, is not considered recombinant. As used herein, the term engineered is synonymous with the term recombinant.
[0061] As used herein, with respect to a meganuclease, the term wild-type refers to any naturally-occurring form of a meganuclease. The term wild-type is not intended to mean the most common allelic variant of the enzyme in nature but, rather, any allelic variant found in nature. Wild-type homing endonucleases are distinguished from recombinant or non-naturally-occurring meganucleases.
[0062] As used herein, the term recognition sequence refers to a DNA sequence that is bound and cleaved by a meganuclease. A recognition sequence comprises a pair of inverted, 9 base pair half sites which are separated by four base pairs. In the case of a homo- or heterodimeric meganucleases, each of the two monomers makes base-specific contacts with one half-site. In the case of a single-chain heterodimer meganuclease, the N-terminal domain of the protein contacts a first half-site and the C-terminal domain of the protein contacts a second half-site. In the case if I-CreI, for example, the recognition sequence is 22 base pairs and comprises a pair of inverted, 9 base pair half sites which are separated by four base pairs.
[0063] As used herein, the term target site refers to a region of the chromosomal DNA of a cell comprising a target sequence into which a sequence of interest can be inserted. As used herein, the term engineered target site refers to an exogenous sequence of DNA integrated into the chromosomal DNA of a cell comprising an engineered target sequence into which a sequence of interest can be inserted.
[0064] As used herein, the term target sequence means a DNA sequence within a target site which includes one or more recognition sequences for a nuclease, integrase, transposase, and/or recombinase. For example, a target sequence can include a recognition sequence for a meganuclease. As used herein, an engineered target sequence means an exogenous target sequence which is introduced into a chromosome to serve as the insertion point for another sequence.
[0065] As used herein, the term flanking region or flanking sequence refers to a sequence of >3 or, preferably, >50 or, more preferably, >200 or, most preferably, >400 base pairs of DNA which is immediately 5 or 3 to a reference sequence (e.g., a target sequence or sequence of interest).
[0066] As used herein, the terms amplifiable locus refers to a region of the chromosomal DNA of a cell which can be amplified by selection with one or more compounds (e.g., drugs) in the growth media. An amplifiable locus will typically comprise a gene encoding a protein which, under the appropriate conditions, is necessary for cell survival. By inhibiting the function of such an essential protein, for example with a small molecule drug, the amplifiable locus is duplicated many times over as a means of increasing the copy number of the essential gene. A gene of interest, if integrated into an amplifiable locus, will also become duplicated with the essential gene. Examples of amplifiable loci include the chromosomal regions comprising the DHFR, GS, and HPRT genes.
[0067] As used herein, the term amplified locus or amplified gene or amplified sequence refers to a locus, gene or sequence which is present in 2-1,000 copies as a result of gene amplification in response to selection of a selectable gene. An amplified gene or sequence can be a gene or sequence which is co-amplified due to selection of a selectable gene in the same amplifiable locus. In preferred embodiments, a sequence of interest is amplified to at least 3, 4, 5, 6, 7, 8, 9 or 10 copies.
[0068] As used herein, the term selectable gene refers to an endogenous gene that is essential for cell survival under some specific culture conditions (e.g., presence or absence of a nutrient, toxin or drug). Selectable genes are endogenous to the cell and are distinguished from exogenous selectable markers such as antibiotic resistance genes. Selectable genes exist in their natural context in the chromosomal DNA of the cell. For example, DHFR is a selectable gene which is necessary for cell survival in the presence of MTX in the culture medium. The gene is essential for growth in the absence of hypoxanthine and thymidine. If the endogenous DHFR selectable gene is eliminated, cells are able to grow in the absence of hypoxanthine and thymidine if they are given an exogenous copy of the DHFR gene. This exogenous copy of the DHFR gene is a selectable marker but is not a selectable gene. An amplifiable locus comprises a selectable gene and a target site. A target site is found outside of a selectable gene such that a selectable gene does not comprise a target site. Examples of selectable genes are given in Table 1.
[0069] As used herein, when used in connection with the position of a target site, recognition sequence, or inserted sequence of interest relative to the position of a selectable gene, the term proximal means that the target site, recognition sequence, or inserted sequence of interest is within the same amplifiable locus as the selectable gene, either upstream (5) or downstream (3) of the selectable gene, and preferably between the selectable gene and the next gene in the region (whether upstream (5) or downstream (3)). Typically, a proximal target site, recognition sequence, or inserted sequence of interest will be within <100,000 base pairs of the selectable gene, as measured from the first or last nucleotide of the first or last regulatory element of the selectable gene.
[0070] As used herein, the term homologous recombination refers to the natural, cellular process in which a double-stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). The homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that was delivered to the cell. Thus, for some applications of engineered meganucleases, a meganuclease is used to cleave a recognition sequence within a target sequence in a genome and an exogenous nucleic acid with homology to or substantial sequence similarity with the target sequence is delivered into the cell and used as a template for repair by homologous recombination. The DNA sequence of the exogenous nucleic acid, which may differ significantly from the target sequence, is thereby inserted or incorporated into the chromosomal sequence. The process of homologous recombination occurs primarily in eukaryotic organisms. The term homology is used herein as equivalent to sequence similarity and is not intended to require identity by descent or phylogenetic relatedness.
[0071] As used herein, the term stably integrated means that an exogenous or heterologous DNA sequence has been covalently inserted into a chromosome (e.g., by homologous recombination, non-homologous end joining, transposition, etc.) and has remained in the chromosome for a period of at least 8 weeks.&&
[0072] As used herein, the term non-homologous end-joining or NHEJ refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non-homologous DNA segments (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA repair by non-homologous end-joining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair. Thus, for certain applications, an engineered meganuclease can be used to produce a double-stranded break at a meganuclease recognition sequence within an amplifiable locus and an exogenous nucleic acid molecule, such as a PCR product, can be captured at the site of the DNA break by NHEJ (see, e.g. Salomon et al. (1998), EMBO J. 17:6086-6095). In such cases, the exogenous nucleic acid may or may not have homology to the target sequence. The process of non-homologous end-joining occurs in both eukaryotes and prokaryotes such as bacteria.
[0073] As used herein, the term sequence of interest means any nucleic acid sequence, whether it codes for a protein, RNA, or regulatory element (e.g., an enhancer, silencer, or promoter sequence), that can be inserted into a genome or used to replace a genomic DNA sequence. Sequences of interest can have heterologous DNA sequences that allow for tagging a protein or RNA that is expressed from the sequence of interest. For instance, a protein can be tagged with tags including, but not limited to, an epitope (e.g., c-myc, FLAG) or other ligand (e.g., poly-His). Furthermore, a sequence of interest can encode a fusion protein, according to techniques known in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, Wiley 1999). In preferred embodiments, a sequence of interest comprises a promoter operably linked to a gene encoding a protein of medicinal value such as an antibody, antibody fragment, cytokine, growth factor, hormone, or enzyme. For some applications, the sequence of interest is flanked by a DNA sequence that is recognized by the engineered meganuclease for cleavage. Thus, the flanking sequences are cleaved allowing for proper insertion of the sequence of interest into genomic recognition sequences cleaved by an engineered meganuclease. For some applications, the sequence of interest is flanked by DNA sequences with homology to or substantial sequence similarity with the target site such that homologous recombination inserts the sequence of interest within the genome at the locus of the target sequence.
[0074] As used herein, the term donor DNA refers to a DNA molecule comprising a sequence of interest flanked by DNA sequences homologous to a target site. Donor DNA can serve as a template for DNA repair by homologous recombination if it is delivered to a cell with a site-specific nuclease such as a meganuclease, zinc-finger nuclease, or TAL-effector nuclease. The result of such DNA repair is the insertion of the sequence of interest into the chromosomal DNA of the cell. Donor DNA can be linear, such as a PCR product, or circular, such as a plasmid. In cases where a donor DNA is a circular plasmid, it may be referred to as a donor plasmid.
[0075] As used herein, unless specifically indicated otherwise, the word or is used in the inclusive sense of and/or and not the exclusive sense of either/or.
2.1 Transgene Targeting to Amplifiable Loci
[0076] The present invention provides methods for generating transgenic mammalian cell lines expressing a desired protein product of interest, including high-producer cell lines, by targeting the insertion of a gene encoding the protein product of interest (e.g., a therapeutic protein gene expression cassette) to regions of the genome that are amplifiable. Such regions in mammalian cells include the DHFR, GS, and HPRT genes, as well as others shown in Table 1.
[0077] The precise mechanism of gene amplification is not known. Indeed, it is very likely that there is no single mechanism by which gene amplification occurs but that a variety of different random chromosomal aberrations, in combination with strong selection for amplification, results in increased gene copy number (reviewed in Omasa (2002), J. Biosci. Bioeng. 94:600-605). It is clear that chromosomal location plays a major role in amplification and the stable maintenance of amplified genes (Brinton and Heintz (1995), Chromosoma 104:143-51). It has been found that transgenes integrated into chromosomal locations adjacent to telomeres are more easily amplified and, once amplified, tend to be stable at high copy numbers after the selection agent is removed (Yoshikawa et al. (2000), Cytotechnology 33:37-46; Yoshikawa et al. (2000), Biotechnol Prog. 16:710-715). This is significant because selection agents such as MTX and MSX are toxic and cannot be included in the growth media in a commercial biomanufacturing process. In contrast, transgenes integrated into regions in the CHO genome that are not adjacent to telomeres amplify inefficiently and rapidly lose copy number following the removal of selection agents from the media. For example, Yoshikawa et al. found that randomly-integrated transgenes linked to a DHFR selectable marker amplified to greater than 10-fold higher copy numbers when the integration site was adjacent to a telomere (Yoshikawa et al. (2000), Biotechnol Prog. 16:710-715). These researchers also found that an amplified transgene integrated into a non-telomeric region will lose >50% of its copies in only 20 days following the removal of MTX from the growth media. None of the selectable genes identified in Table 1 is adjacent to a telomere in the mouse genome (www.ensembl.com) and the similarity in genome organization between mouse and CHO makes it likely that these genes are in non-telomeric regions in CHO as well (Xu et al. (2011), Nat. Biotechnol. 29:735-741). Thus, the prior art instructs that the loci identified in Table 1, including the DHFR and GS loci, are not preferred locations to target transgene insertion if the goal is efficient and stable gene amplification.
[0078] In addition, in the case of endogenous gene amplification, it is clear that chromosomal sequences outside of the selectable gene sequence play an important role in facilitating amplification and in defining the length of DNA sequence that is co-amplified with the gene under selection (Looney and Hamlin (1987), Mol. and Cell. Biol. 7:569-577). In particular, it has been shown that the sequence and location of the DNA replication origin in relation to the selectable gene plays a major role in amplification. For example, it has been shown that amplification of the endogenous CHO DHFR locus is dependent upon a pair of replication origins found in the region 5,000-60,000 base pairs downstream of the DHFR gene coding sequence (Anachkova and Hamlin (1989), Mol. and Cell. Biol. 9:532-540; Milbrandt et al. (1981), Proc. Natl. Acad. Sci. USA 78:6042-6047). Further, Brinton and Heintz have shown that these same replication origins fail to promote gene amplification when incorporated randomly into the genome with a transgenic DHFR sequence (Brinton and Heintz (1995), Chromosoma. 104:143-51). This clearly demonstrates the importance of maintaining both the sequence and proper chromosomal context of these replication origins to promote DHFR gene amplification. Thus the art instructs that the region downstream of DHFR is critical to gene amplification and should not be disrupted by, for example, inserting a transgenic gene expression cassette as described in the present invention.
[0079] Surprisingly, we have discovered that DNA sequences, including exogenous transcriptionally active sequences, which are inserted proximal to (e.g., within <100,000 base pairs) selectable genes in mammalian cell lines (e.g., CHO-K1) will co-amplify in the presence of appropriate compounds which select for amplification. Thus, the present invention provides methods for reliably and reproducibly producing isogenic cell lines in which transgenes encoding protein products of interest (e.g., biotherapeutic gene expression cassettes) can be amplified but in which it is not necessary to screen a large number of randomly generated cell lines to identify those which express high levels of the protein product of interest and are resistant to gene silencing.
[0080] In addition, we have surprisingly found that the mammalian cell lines of the invention, in which a sequence of interest is co-amplified with a selectable gene in an amplifiable locus, are stable with respect to expression of the sequence of interest and/or copy number of the sequence of interest even in the absence of continued selection. That is, whereas the art teaches that amplified sequences will be reduced in copy number over time if selection is not maintained (see, e.g., Yoshikawa et al. (2000), Biotechnol Prog. 16:710-715), we have found that cell lines produced according to the methods of the invention continue to produce the protein products of interest (encoded by the sequences of interest) at levels within 20%-25% of the initial levels, even 14 weeks after removal of the selection agent. This is significant, as noted above, because selection agents such as MTX and MSX are toxic, and it would be highly desirable to produce biotherapeutic proteins in cell lines which do not require continued exposure to such selection agents. Therefore, in some embodiments, the invention provides recombinant mammalian cell lines which continue to express a protein product of interest from an exogenous sequence of interest present in an amplified region of the genome (i.e., present in 2-1,000 copies, co-amplified with a selectable gene in an amplifiable locus) for a period of at least 8, 9, 10, 11, 12, 13, or 14 weeks after removal of the amplification selection agent, and with a reduction of expression levels and/or copy number of less than 20, 25, 30, 35 or 40%.
[0081] The present invention also provides the products necessary to practice the methods, and to target insertion of sequences of interest into amplifiable loci in mammalian cell lines. A common method for inserting or modifying a DNA sequence involves introducing a transgenic DNA sequence flanked by sequences homologous to the genomic target and selecting or screening for a successful homologous recombination event. Recombination with the transgenic DNA occurs rarely but can be stimulated by a double-stranded break in the genomic DNA at the target site (Porteus et al. (2005), Nat. Biotechnol. 23: 967-73; Tzfira et al. (2005), Trends Biotechnol. 23: 567-9; McDaniel et al. (2005), Curr. Opin. Biotechnol. 16: 476-83). Numerous methods have been employed to create DNA double-stranded breaks, including irradiation and chemical treatments. Although these methods efficiently stimulate recombination, the double-stranded breaks are randomly dispersed in the genome, which can be highly mutagenic and toxic. At present, the inability to target gene modifications to unique sites within a chromosomal background is a major impediment to routine genome engineering.
[0082] One approach to achieving this goal is stimulating homologous recombination at a double-stranded break in a target locus using a nuclease with specificity for a sequence that is sufficiently large to be present at only a single site within the genome (see, e.g., Porteus et al. (2005), Nat. Biotechnol. 23: 967-73). The effectiveness of this strategy has been demonstrated in a variety of organisms using ZFNs (Porteus (2006), Mol Ther 13: 438-46; Wright et al. (2005), Plant J. 44: 693-705; Urnov et al. (2005), Nature 435: 646-51). Homing endonucleases are a group of naturally-occurring nucleases which recognize 15-40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as Group I self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double-stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Homing endonucleases are commonly grouped into four families: the LAGLIDADG (SEQ ID NO: 65) family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLIDADG (SEQ ID NO: 65) family are characterized by having either one or two copies of the conserved LAGLIDADG (SEQ ID NO: 65) motif (see Chevalier et al. (2001), Nucleic Acids Res. 29(18): 3757-3774). The LAGLIDADG (SEQ ID NO: 65) homing endonucleases with a single copy of the LAGLIDADG (SEQ ID NO: 65) motif form homodimers, whereas members with two copies of the LAGLIDADG (SEQ ID NO: 65) motif are found as monomers.
[0083] Natural homing endonucleases, primarily from the LAGLIDADG (SEQ ID NO: 65) family, have been used to effectively promote site-specific genome modification in plants, yeast, Drosophila, mammalian cells and mice, but this approach has been limited to the modification of either homologous genes that conserve the endonuclease recognition sequence (Monnat et al. (1999), Biochem. Biophys. Res. Commun. 255: 88-93) or to pre-engineered genomes into which a recognition sequence has been introduced (Rouet et al. (1994), Mol. Cell. Biol. 14: 8096-106; Chilton et al. (2003), Plant Physiol. 133: 956-65; Puchta et al. (1996), Proc. Natl. Acad. Sci. USA 93: 5055-60; Rong et al. (2002), Genes Dev. 16: 1568-81; Gouble et al. (2006), J. Gene Med. 8(5):616-622).
[0084] Systematic implementation of nuclease-stimulated gene modification requires the use of engineered enzymes with customized specificities to target DNA breaks to existing sites in a genome and, therefore, there has been great interest in adapting homing endonucleases to promote gene modifications at medically or biotechnologically relevant sites (Porteus et al. (2005), Nat. Biotechnol. 23: 967-73; Sussman et al. (2004), J. Mol. Biol. 342: 31-41; Epinat et al. (2003), Nucleic Acids Res. 31: 2952-62).
[0085] I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG (SEQ ID NO: 65) family of homing endonucleases which recognizes and cuts a 22 base pair recognition sequence in the chloroplast chromosome of the algae Chlamydomonas reinhardtii. Genetic selection techniques have been used to modify the wild-type I-CreI cleavage site preference (Sussman et al. (2004), J. Mol. Biol. 342: 31-41; Chames et al. (2005), Nucleic Acids Res. 33: e178; Seligman et al. (2002), Nucleic Acids Res. 30: 3870-9, Arnould et al. (2006), J. Mol. Biol. 355: 443-58). More recently, a method of rationally-designing mono-LAGLIDADG (SEQ ID NO: 65) homing endonucleases was described which is capable of comprehensively redesigning I-CreI and other homing endonucleases to target widely-divergent DNA sites, including sites in mammalian, yeast, plant, bacterial, and viral genomes (WO 2007/047859).
[0086] Thus, in one embodiment, the invention provides engineered meganucleases derived from the amino acid sequence of I-CreI that recognize and cut DNA sites in amplifiable regions of mammalian genomes. These engineered meganucleases can be used in accordance with the invention to target the insertion of gene expression cassettes into defined locations in the chromosomal DNA of cell lines such as CHO cells. This invention will greatly streamline the production of desired cell lines by reducing the number of lines that must be screened to identify a high-producer clone suitable for commercial-scale production of a therapeutic glycoprotein.
[0087] The present invention involves targeting transgenic DNA sequences of interest to amplifiable loci. The amplifiable loci are regions of the chromosomal DNA that contain selectable genes that become amplified in the presence of selection agents (e.g., drugs). For example, the Chinese Hamster Ovary (CHO) cell DHFR locus can be amplified to 1,000 copies by growing the cells in the presence of methotrexate (MTX), a DHFR inhibitor. Table 1 lists additional examples of selectable genes that can be amplified using small molecule drugs (Kellems, ed. Gene amplification in mammalian cells: a comprehensive guide. Marcel Dekker, New York, 1993; Omasa (2002), J. Biosci. Bioeng. 94:6 600-605).
TABLE-US-00001 TABLE 1 Amplifiable Genes Selectable Gene Name Amplified With Dihydrofolate Reductase Methotrexate (MTX) Glutamine Synthetase Methionine sulphoximine (MSX) Hypoxanthine Phosphoribosyl- Aminopterin, hypoxanthine, and thymidine transferase Threonyl tRNA Synthetase Borrelidin Na,K-ATPase Ouabain Asparagine Synthetase Albizziin or Beta-aspartyl hydroxamate Ornithine Decarboxylase alpha-difluoromethylornithine (DFMO) Inosine-5-monophosphate Mycophenolic Acid dehydrogenase Adenosine Deaminase Adenosine, Alanosine, 2deoxycoformycin Thymidylate Synthetase Fluorouracil Aspartate Transcarbamylase N-Phosphonacetyl-L-Aspartate (PALA) Metallothionein Cadmium Adenylate Deaminase (1, 2) Adenine, Azaserine, Coformycin UMP-Synthetase 6-azauridine, pyrazofuran Ribonucleotide Reductase hydroxyurea, motexafin gadolinium, fludarabine, cladribine, gemcitabine, tezacitabine, triapine.
[0088] Several considerations must be taken into account when selecting a specific target site for the insertion of a sequence of interest within an amplifiable locus. First, the selected insertion site must be co-amplified with the gene under selection. In many cases, experimental data already exists in the art which delimits the amount of flanking chromosomal sequence that co-amplifies with a selectable gene of interest. This data, which precisely defines the extent of the amplifiable locus, exists for CHO DHFR (Ma et al. (1988), Mol Cell Biol. 8(6):2316-27), human DHFR (Morales et al. (2009), Mol Cancer Ther. 8(2):424-432), and CHO GS (Sanders et al. (1987), Dev Biol Stand. 66:55-63). Where such data does not already exist in the art, we predict that chromosomal DNA sequences <100,000 base pairs upstream or downstream of the selectable gene coding sequence are likely to co-amplify. Hence, these regions could be suitable sites for targeting the insertion of a sequence of interest.
[0089] Second, target sites should be selected which will not greatly impact the function of the selectable gene (e.g., the endogenous DHFR, GS, or HPRT gene). Because amplification requires a functional copy of the selectable gene, insertion sites within the promoter, exons, introns, polyadenylation signals, or other regulatory sequences that, if disrupted, would greatly impact transcription or translation of the selectable gene, should be avoided. For example, WO 2008/059317 discloses meganucleases which cleave DNA target sites within the HPRT gene. To the extent WO 2008/059317 discloses the insertion of genes into the HPRT locus, it teaches that the HPRT gene coding sequence should be disrupted in the process of transgene insertion to facilitate selection for proper targeting using 6-thioguanine. 6-thioguanine is a toxic nucleotide analog that kills cells having functional HPRT activity. Because cells produced in accordance with WO 2008/059317 will not have HPRT activity, they will not amplify an inserted transgene in response to treatment with an HPRT inhibitor and, so, cannot be used in the present invention. For the present invention, unless the precise limits of all regulatory sequences are already known for a particular selectable gene, insertion sites >1,000 base pairs, >2,000 base pairs, >3,000 base pairs, >4,000 base pairs, or, preferably, >5,000 base pairs, upstream or downstream of the gene coding sequence should be selected. However, if the location of the regulatory sequences are known, the sequence of interest can be inserted immediately adjacent to the either the most 5 or 3 regulatory sequence (e.g., immediately 3 to the polyadenylation signal).
[0090] Lastly, target sites should be selected which do not disrupt other chromosomal genes which may be important for normal cell physiology. In general, gene insertion sites should be >1,000 base pairs, >2,000 base pairs, >3,000 base pairs, >4,000 base pairs, or, preferably, >5,000 base pairs, away from any gene coding sequence.
[0091] Various methods of the invention are described schematically in the figures as follows:
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2.1.1 Gene Targeting to the CHO DHFR Locus
[0107] The CHO DHFR locus is diagrammed in
[0108] The human and mouse DHFR loci have an organization similar to CHO locus. In both cases, the Msh3 gene is immediately upstream of DHFR but there is a large area devoid of coding sequences downstream of DHFR. In humans, the ANKRD34B gene is 55,000 base pairs downstream of DHFR while the ANKRD34B gene is 37,000 base pairs downstream of DHFR in mouse. Therefore, the genomic region downstream of DHFR is an appropriate location to insert genes of interest in CHO, human, and mouse cells and cell lines. Further, gene expression cassettes inserted into this region will be expressed at a high level, resistant to gene silencing, and capable of being amplified by treatment with MTX. Methods for amplifying the CHO cell DHFR locus are known in the art (see, e.g., Kellems, ed., Gene amplification in mammalian cells: a comprehensive guide. Marcel Dekker, New York, 1993) and typically involve gradually increasing the concentration of MTX in the growth media from 0 to as high as 0.8 mM over a period of several weeks.
2.1.2 Gene Targeting to the GS Locus
[0109] The CHO, human, and mouse glutamine synthetase (also known as glutamate-ammonia ligase or GluL) loci share a common organization (
2.2 Engineered Endonucleases for Gene Targeting
[0110] A sequence of interest may be inserted into an amplifiable locus using an engineered site-specific endonuclease. Methods for generating site-specific endonucleases which can target DNA breaks to pre-determined loci in a genome are known in the art. These include zinc-finger nucleases (Le Provost et al. (2010), Trends Biotechnol. 28(3):134-41), TAL-effector nucleases (Li et al. (2011), Nucleic Acids Res. 39(1):359-72), and engineered meganucleases (WO 2007/047859; WO 2007/049156; WO 2009/059195). In one embodiment, the invention provides engineered meganucleases derived from I-CreI that can be used to target the insertion of a gene of interest to an amplifiable locus. Methods to produce such engineered meganucleases are known in the art (see, e.g., WO 2007/047859; WO 2007/049156; WO 2009/059195). In preferred embodiments, a single-chain meganuclease is used to target gene insertion to an amplifiable region of the genome. Methods for producing such single-chain meganucleases are known in the art (see, e.g., WO 2009/059195 and WO 2009/095742). In some embodiments, the engineered nuclease is fused to a nuclear localization signal (NLS) to facilitate nuclear uptake. Examples of nuclear localization signals include the SV40 NLS (amino acid sequence MAPKKKRKV (SEQ ID NO: 36)) which can be fused to the C- or, preferably, the N-terminus of the protein. In addition, an engineered nuclease may be tagged with a peptide epitope (e.g., an HA, FLAG, or Myc epitope) to monitor expression levels or localization or to facilitate purification.
2.3 Engineered Cell Lines with Sequences of Interest Targeted to Amplifiable Loci
[0111] In some embodiments, the invention provides methods for using engineered nucleases to target the insertion of transgenes into amplifiable loci in cultured mammalian cells. This method has two primary components: (1) an engineered nuclease; and (2) a donor DNA molecule comprising a sequence of interest. The method comprises contacting the DNA of the cell with the engineered nuclease to create a double strand DNA break in an endogenous recognition sequence in an amplifiable locus followed by the insertion of the donor DNA molecule at the site of the DNA break. Such insertion of the donor DNA is facilitated by the cellular DNA-repair machinery and can occur by either the non-homologous end-joining pathway or by homologous recombination (
[0112] The engineered nuclease can be delivered to the cell in the form protein or, preferably, as a nucleic acid encoding the engineered nuclease. Such nucleic acid can be DNA (e.g., circular or linearized plasmid DNA or PCR products) or RNA. For embodiments in which the engineered nuclease coding sequence is delivered in DNA form, it should be operably linked to a promoter to facilitate transcription of the engineered nuclease gene. Mammalian promoters suitable for the invention include constitutive promoters such as the cytomegalovirus early (CMV) promoter (Thomsen et al. (1984), Proc Natl Acad Sci USA. 81(3):659-63) or the SV40 early promoter (Benoist and Chambon (1981), Nature. 290(5804):304-10) as well as inducible promoters such as the tetracycline-inducible promoter (Dingermann et al. (1992), Mol Cell Biol. 12(9):4038-45).
[0113] In some embodiments, mRNA encoding the engineered nuclease is delivered to the cell because this reduces the likelihood that the gene encoding the engineered nuclease will integrate into the genome of the cell. Such mRNA encoding an engineered nuclease can be produced using methods known in the art such as in vitro transcription. In some embodiments, the mRNA is capped using 7-methyl-guanosine. In some embodiments, the mRNA may be polyadenylated.
[0114] Purified engineered nuclease proteins can be delivered into cells to cleave genomic DNA, which allows for homologous recombination or non-homologous end-joining at the cleavage site with a sequence of interest, by a variety of different mechanisms known in the art. For example, the recombinant nuclease protein can be introduced into a cell by techniques including, but not limited to, microinjection or liposome transfections (see, e.g., Lipofectamine, Invitrogen Corp., Carlsbad, Calif.). The liposome formulation can be used to facilitate lipid bilayer fusion with a target cell, thereby allowing the contents of the liposome or proteins associated with its surface to be brought into the cell. Alternatively, the enzyme can be fused to an appropriate uptake peptide such as that from the HIV TAT protein to direct cellular uptake (see, e.g., Hudecz et al. (2005), Med. Res. Rev. 25: 679-736).
[0115] Alternatively, gene sequences encoding the engineered nuclease protein are inserted into a vector and transfected into a eukaryotic cell using techniques known in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, Wiley 1999). The sequence of interest can be introduced in the same vector, a different vector, or by other means known in the art. Non-limiting examples of vectors for DNA transfection include virus vectors, plasmids, cosmids, and YAC vectors. Transfection of DNA sequences can be accomplished by a variety of methods known to those of skill in the art. For instance, liposomes and immunoliposomes are used to deliver DNA sequences to cells (see, e.g., Lasic et al. (1995), Science 267: 1275-76). In addition, viruses can be utilized to introduce vectors into cells (see, e.g., U.S. Pat. No. 7,037,492). Alternatively, transfection strategies can be utilized such that the vectors are introduced as naked DNA (see, e.g., Rui et al. (2002), Life Sci. 71(15): 1771-8).
[0116] General methods for delivering nucleic acids into cells include: (1) chemical methods (Graham et al. (1973), Virology 54(2):536-539; Zatloukal et al. (1992), Ann. N.Y. Acad. Sci., 660:136-153; (2) physical methods such as microinjection (Capecchi (1980), Cell 22(2):479-488, electroporation (Wong et al. (1982), Biochim. Biophys. Res. Commun. 107(2):584-587; Fromm et al. (1985), Proc. Nat'l Acad. Sci. USA 82(17):5824-5828; U.S. Pat. No. 5,384,253) and ballistic injection (Johnston et al. (1994), Methods Cell. Biol. 43(A): 353-365; Fynan et al. (1993), Proc. Nat'l Acad. Sci. USA 90(24): 11478-11482); (3) viral vectors (Clapp (1993), Clin. Perinatol. 20(1): 155-168; Lu et al. (1993), J. Exp. Med. 178(6):2089-2096; Eglitis et al. (1988), Avd. Exp. Med. Biol. 241:19-27; Eglitis et al. (1988), Biotechniques 6(7):608-614); and (4) receptor-mediated mechanisms (Curiel et al. (1991), Proc. Nat'l Acad. Sci. USA 88(19):8850-8854; Curiel et al. (1992), Hum. Gen. Ther. 3(2):147-154; Wagner et al. (1992), Proc. Nat'l Acad. Sci. USA 89 (13):6099-6103). In some preferred embodiments, 7-methyl-guanosine capped mRNA encoding the engineered nuclease is delivered to cells using electroporation.
[0117] The donor DNA molecule comprises a gene of interest operably linked to a promoter. In many cases, a donor molecule may comprise multiple genes operably linked to the same or different promoters. For example, donor molecules comprising monoclonal antibody expression cassettes may comprise a gene encoding the antibody heavy chain and a second gene encoding the antibody light chain. Both genes may be under the control of different promoters or they may be under the control of the same promoter by using, for example, an internal-ribosome entry site (IRES). Donor molecules may also comprise a selectable marker gene operably linked to a promoter to facilitate the identification of transgenic cells. Such selectable markers are known in the art and include neomycin phosphotransferase (NEO), hypoxanthine phosphoribosyltransferase (HPRT), glutamine synthetase (GS), dihydrofolate reductase (DHFR), and hygromycin phosphotransferase (HYG) genes.
[0118] In some embodiments, donor DNA molecules will additionally comprise flanking sequences homologous to the target sequences in the DNA of the cell. Such homologous flanking sequences comprise >3 or, preferably, >50 or, more preferably, >200 or, most preferably, >400 base pairs of DNA that are identical or nearly identical in sequence to the chromosomal locus recognized by the engineered nuclease (
[0119] The donor DNA molecule can be circular (e.g., plasmid DNA) or linear (e.g., linearized plasmid or PCR products). Methods for delivering DNA molecules are known in the art, as discussed above.
[0120] In some embodiments, the engineered nuclease gene and donor DNA are carried on separate nucleic acid molecules which are co-transfected into cells or cell lines. For example, the engineered nuclease gene operably linked to a promoter can be transfected in plasmid form simultaneously with a separate donor DNA molecule in plasmid or PCR product form. In an alternative embodiment, the engineered nuclease can be delivered in mRNA form with a separate donor DNA molecule in plasmid or PCR product form. In a third embodiment, the engineered nuclease gene and donor DNA are carried on the same DNA molecule, such as a plasmid. In a fourth embodiment, cells are co-transfected with purified engineered nuclease protein and a donor DNA molecule in plasmid or PCR product form.
[0121] Following transfection with the engineered nuclease and donor DNA, cells are typically allowed to recover from transfection (24-72 hours) before being cloned using methods known in the art. Common methods for cloning a genetically engineered cell line include limiting dilution in which transfected cells are transferred to tissue culture plates (e.g., 48 well, 96 well plates) at a concentration of <1 cell per well and expanded into clonal populations. Other cloning strategies include robotic clone identification/isolation systems such as ClonePix (Genetix, Molecular Devices, Inc., Sunnyvale, Calif.). Clonal cell lines can then be screened to identify cell lines in which the sequence of interest is integrated into the intended target site. Cell lines can easily be screened using molecular analyses known in the art such as PCR or Southern Blot. For example, genomic DNA can be isolated from a clonal cell line and subjected to PCR amplification using a first (sense-strand) primer that anneals to a DNA sequence in the sequence of interest and a second (anti-sense strand) primer that anneals to a sequence in the amplifiable locus. If the donor DNA molecule comprises a DNA sequence homologous to the target site, it is important that the second primer is designed to anneal to a sequence in the amplifiable locus that is beyond the limits of homology carried on the donor molecule to avoid false positive results. Alternatively, cell lines can be screened for expression of the sequence of interest. For example, if the sequence of interest encodes a secreted protein such as an antibody, the growth media can be sampled from isolated clonal cell lines and assayed for the presence of antibody protein using methods known in the art such as Western Blot or Enzyme-Linked Immunosorbant Assay (ELISA). This type of functional screen can be used to identify clonal cell lines which carry at least one copy of the sequence of interest integrated into the genome. Additional molecular analyses such as PCR or Southern blot can then be used to determine which of these transgenic cell lines carry the sequence of interest targeted to the amplifiable locus of interest, as described above.
[0122] The method of the invention can be used on any culturable and transfectable cell type such as immortalized cell lines and stem cells. In preferred embodiments, the method of the invention is used to genetically modify immortalized cell lines that are commonly used for biomanufacturing. This includes: [0123] 1. Hamster cell lines such as baby hamster kidney (BHK) cells and all variants of Chinese Hamster Ovary (CHO) cells, e.g., CHO-K1, CHO-S (Invitrogen Corp., Carlsbad, Calif.), DG44, or Potelligent (Lonza Group Ltd., Basel, Switzerland). Because the genome sequences of different hamster cell lines are very nearly identical, an engineered meganuclease which can be used to practice the invention in one hamster cell type (e.g., BHK cells) can generally be used to practice the invention in another hamster cell type (e.g., CHO-K1). [0124] 2. Mouse cell lines such as mouse hybridoma or mouse myeloma (e.g., NS0) cells. Because the genome sequences of different mouse cell lines are very nearly identical, an engineered meganuclease which can be used to practice the invention in one mouse cell type (e.g., mouse hybridoma cells) can generally be used to practice the invention in another mouse cell type (e.g., NS0). [0125] 3. Human cell lines such as human embryonic kidney cells (e.g., HEK-293 or 293S) and human retinal cells (e.g., PER.C6). Because the genome sequences of different human cell lines are very nearly identical, an engineered meganuclease which can be used to practice the invention in one human cell type (e.g., HEK-293 cells) can generally be used to practice the invention in another human cell type (e.g., PER.C6).
2.6 Pre-Engineered Cell Lines with Engineered Target Sequences in Amplifiable Loci
[0126] In one embodiment, the invention provides cell lines which are pre-engineered to comprise a targetable engineered target sequence for gene insertion in an amplifiable locus in a mammalian cell line (
[0127] In an alternative embodiment, a cell line is produced by inserting an engineered target sequence into an amplifiable locus with the concomitant removal of all or a portion of the adjacent endogenous marker gene (
[0128] In an alternative embodiment, a cell line is produced in which an engineered target sequence is inserted into an amplifiable locus with disruption of the selectable gene (
2.5 Transgenic Cell Lines for Biomanufacturing
[0129] In some embodiments, the invention provides transgenic cell lines suitable for the production of protein pharmaceuticals. Such transgenic cell lines comprise a population of cells in which a gene of interest, operably linked to a promoter, is inserted into the genome of the cell at an amplifiable locus wherein the gene of interest encodes a protein therapeutic. Examples of protein therapeutics include: monoclonal antibodies, antibody fragments, erythropoietin, tissue-type plasminogen activator, Factor VIII, Factor IX, insulin, colony stimulating factors, interferons (e.g., interferon-, interferon-, and interferon-), interleukins (e.g., interleukin-2), vaccines, tumor necrosis factor, and glucocerebrosidase. Protein therapeutics are also referred to as biologics or biopharmaceuticals.
[0130] To be used for biomanufacturing, a transgenic cell line of the invention should undergo: (1) adaptation to serum-free growth in suspension; and (2) amplification of the gene of interest. In some embodiments, the invention is practiced on adherent cell lines which can be adapted to growth in suspension to facilitate their maintenance in shaker-flasks or stirred-tank bioreactors as is typical of industrial biomanufacturing. Methods for adapting adherent cells to growth in suspension are known in the art (Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). For regulatory reasons, it is generally necessary to further adapt biomanufacturing cell lines to chemically-defined media lacking animal-derived components (i.e., serum-free media). Methods for preparing such media and adapting cell lines to it are known in the art (Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). Such media can also be purchased commercially (e.g., CD-3 media for maintenance of CHO cells, available from Sigma-Aldrich, St. Louis, Mo.) and cells can be adapted to it by following the manufacturers' instructions. In some embodiments, the cell line is adapted to growth in suspension and/or serum-free media prior to being transfected with the engineered nuclease.
[0131] Lastly, methods for gene amplification are known in the art (Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). In general, the process involves adding an inhibitor of a selectable gene product to the growth media to select for cells that express abnormally high amounts of the gene product due to gene-duplication events. In general, the concentration of inhibitor added to the growth media is increased slowly over a period of weeks until the desired level of gene amplification is achieved. Inhibitor is then generally removed from the media prior to initiating a bioproduction run to avoid the possibility of the inhibitor contaminating the protein therapeutic formulation. For example, the CHO DHFR locus can be amplified by slowly increasing the concentration of MTX in the growth media from 0 mM to as high as 0.8 mM over a period of several weeks. The GS locus can, likewise, be amplified by slowly increasing the concentration of MSX in the media from 0 M to as high as 100 M over a period of several weeks. Methods for evaluating gene amplification are known in the art and include Southern Blot and quantitative real-time PCR (rtPCR). In addition, or as an alternative, expression levels of the sequence of interest, which are generally correlated to gene copy number, can be evaluated by determining the concentration of protein therapeutic in the growth media using conventional methods such as Western Blot or ELISA.
[0132] Following cell line production, adaptation, and amplification, protein therapeutics can be produced and purified using methods that are standard in the biopharmaceutical industry.
EXAMPLES
[0133] This invention is further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are intended to be encompassed in the scope of the claims that follow the examples below. Example 1 refers to engineered meganucleases that can be used to target the insertion of a gene of interest downstream of the DHFR gene in CHO cells. Example 2 refers to engineered meganucleases that can be used to target the insertion of an engineered target sequence into the CHO DHFR gene with concomitant removal of DHFR exons 1 and 2. Example 2 also refers to engineered meganucleases that can be used to target the insertion of an engineered target sequence into the CHO GS gene. Example 3 refers to meganucleases that can be used to target the insertion of a gene of interest downstream of the GS gene in CHO cells.
Example 1
[0134] Targeted Gene Insertion into the CHO DHFR Locus Using Engineered Meganucleases
[0135] The CHO genomic DNA sequence 10,000-55,000 base pairs downstream of the DHFR gene was searched to identify DNA sites amenable to targeting with engineered meganucleases. Two sites (SEQ ID NO: 7 and SEQ ID NO: 8) were selected which are, respectively, 35,699 and 15,898 base pairs downstream of the DHFR coding sequence (Table 2).
TABLE-US-00002 TABLE2 ExampleRecognitionSitesForEngineeredMeganucleasesin theCHODHFRLocus. SEQID LocationRelativetoCHO NO: TargetSiteSequences DHFRCodingSequence 7 5-TAAGGCCTCATATGAAAATATA-3 35,699bpdownstream 8 5-ATAGATGTCTTGCATACTCTAG-3 15,898bpdownstream
1. Meganucleases that Recognize SEQ ID NO: 7 and SEQ ID NO: 8
[0136] An engineered meganuclease (SEQ ID NO: 9) was produced which recognizes and cleaves SEQ ID NO: 7. This meganuclease is called CHO-23/24. A second engineered meganuclease (SEQ ID NO: 10) was produced which recognizes and cleaves SEQ ID NO: 8. This meganuclease is called CHO-51/52. Each meganuclease comprises an N-terminal nuclease-localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit.
2. Site-Specific Cleavage of Plasmid DNA by Meganucleases CHO-23/24 and CHO-51/52
[0137] CHO-23/24 and CHO-51/52 were evaluated using a direct-repeat recombination assay as described previously (Gao et al. (2010), Plant J. 61(1):176-87,
[0138] The coding sequences of the engineered meganucleases were inserted into the mammalian expression vector pCP under the control of a constitutive (CMV) promoter. Chinese hamster ovary (CHO) cells at approximately 90% confluence were transfected in 96-well plates with 150 ng pGFFP reporter plasmid and 50 ng of meganuclease expression vector or, to determine background, 50 ng of empty pCP, using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen Corp., Carlsbad, Calif.). To determine transfection efficiency, CHO cells were transfected with 200 ng pCP GFP. Cells were washed in PBS 24 h post-transfection, trypsinized and resuspended in PBS supplemented with 3% fetal bovine serum. Cells were assayed for GFP activity using a Cell Lab Quanta SC MPL flow cytometer and the accompanying Cell Lab Quanta analysis software (Beckman Coulter, Brea, Calif.).
[0139] Results are shown in
3. Site-Specific Cleavage of CHO DHFR Locus by Meganucleases CHO-23/24 and CHO-51/52
[0140] To determine whether or not CHO-23/24 and CHO-51/52 are capable of cleaving their intended target sites in the CHO DHFR locus, we screened genomic DNA from CHO cells expressing either CHO-23/24 or CHO-51/52 to identify evidence of chromosome cleavage at the intended target site. This assay relies on the fact that chromosomal DNA breaks are frequently repaired by NHEJ in a manner that introduces mutations at the site of the DNA break. These mutations, typically small deletions or insertions (collectively known as indels) leave a telltale scar that can be detected by DNA sequencing (Gao et al. (2010), Plant J. 61(1):176-87).
[0141] CHO cells were transfected with mRNA encoding CHO-23/24 or CHO-51/52. mRNA was prepared by first producing a PCR template for an in vitro transcription reaction (SEQ ID NO: 20 and SEQ ID NO: 21). Each PCR product included a T7 promoter and 609 bp of vector sequence downstream of the meganuclease gene. The PCR product was gel purified to ensure a single template. Capped (m7G) RNA was generated using the RiboMAX T7 kit (Promega Corp., Fitchburg, Wis.) according to the manufacturer's instructions and. Ribo m7G cap analog (Promega Corp., Fitchburg, Wis.) was included in the reaction and 0.5 g of the purified meganuclease PCR product served as the DNA template. Capped RNA was purified using the SV Total RNA Isolation System (Promega Corp., Fitchburg, Wis.) according to the manufacturer's instructions.
[0142] 1.510.sup.6CHO-K1 cells were nucleofected with 310.sup.12 copies of CHO-23/24 or CHO-51/52 mRNA (210.sup.6 copies/cell) using an Amaxa Nucleofector II device (Lonza Group Ltd., Basel, Switzerland) and the U-23 program according to the manufacturer's instructions. 48 hours post-transfection, genomic DNA was isolated from the cells using a FlexiGene kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The genomic DNA was then subjected to PCR to amplify the corresponding target site. In the case of cells transfected with mRNA encoding CHO-23/24, the forward and reverse PCR primers were SEQ ID NO: 16 and SEQ ID NO: 17. In the case of cells transfected with mRNA encoding CHO-51/52, the forward and reverse PCR primers were SEQ ID NO: 18 and SEQ ID NO: 19. PCR products were gel purified and cloned into pUC-19. 40 plasmids harboring PCR products derived from cells transfected with CHO-23/24 mRNA were sequenced, 13 of which were found to have mutations in the CHO-23/24 target site (
4. Site-Specific Integration into the CHO DHFR Locus Using an Engineered Meganuclease
[0143] To evaluate the efficiency of DNA insertion into the CHO DHFR locus using an engineered meganuclease, we prepared a donor plasmid (SEQ ID NO: 11) comprising an EcoRI restriction enzyme site flanked by DNA sequence homologous to the CHO-51/52 recognition site (
[0144] mRNA encoding CHO-51/52 was prepared as described above. 1.510.sup.6 CHO-K1 cells were nucleofected with 310.sup.12 copies of CHO 51-52 mRNA (210.sup.6 copies/cell) and 1.5 g of the donor plasmid (SEQ ID NO: 11). Nucleofection was performed using an Amaxa Nucleofector II device (Lonza Group Ltd., Basel, Switzerland) and the U-23 program according to the manufacturer's instructions. 48 hours post-transfection, genomic DNA was isolated from the cells using a FlexiGene kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA was subjected to PCR using primers flanking the CHO-51/52 recognition site (SEQ ID NO: 18 and SEQ ID NO: 19). Importantly, these primers are beyond the limits of homologous sequence carried in the donor plasmid and, therefore, will amplify only the chromosomal DNA sequence and not the donor plasmid. PCR products were cloned into a pUC-19 plasmid and 48 clones were purified and digested with EcoRI (
5. Site-Specific Integration of an Engineered Target Sequence into the CHO DHFR Locus
[0145] A donor plasmid (SEQ ID NO: 25) was produced comprising an FRT sequence (SEQ ID NO: 5) adjacent to a zeocin resistance gene under the control of an SV40 early promoter (
[0146] Hygromycin-resistant cells were cloned by limiting dilution and 24 individual clonal lines were assayed by PCR using a first primer in the hygromycin-resistance gene (SEQ ID NO: 28). All 24 clones yielded the expected PCR product (
6. Transgene Amplification
[0147] A GFP-expressing CHO line produced as described above was seeded at a density of 310.sup.5 cells/mL in 30 mL of media containing 50 nM MTX. Cells were cultured for 14 days before being re-seeded at the same density in media containing 100 nM MTX. Cells were cultured for another 14 days before being re-seeded in media containing 250 nM MTX. Following 14 days in culture, GFP expression in the treated cells was evaluated by flow cytometry and compared to GFP expression in the parental (pre-MTX) cell population (
7. Stability of Gene Amplification
[0148] The five clonal cell lines expressing high levels of GFP that were produced in (6) above were then passaged for a period of 14 weeks in media with or without 250 nM MTX to evaluate the stability of gene amplification. GFP intensity was determined on a weekly basis and the quantitative PCR assay used to determine GFP gene copy number described above was repeated at the end of the 14 week evaluation period. As expected, the clones passaged in media with MTX maintained a high level of GFP expression with no clone deviating more than 20% from the GFP intensity determined in week 1. Quantitative PCR revealed that gene copy number likewise deviated by less than 20% for all clones. Surprisingly, gene amplification was equally stable in cell lines grown in media lacking MTX. Contrary to what would have been predicted based on the existing art, GFP gene expression was not reduced by more than 18% in any of the five cell lines over the 14 week evaluation period. Gene copy number determined by quantitative PCR was also stable with less than 24% deviation over time for all of the cell lines. These results indicate that a transgene amplified in the CHO DHFR locus is stable for an extended period of time, obviating the need to grow the cells in toxic selection agents that that could contaminate bioproduct formulations.
Example 2
Insertion of an Engineered Target Sequence into the CHO DHFR or GS Gene Coding Regions
[0149] As diagrammed in
1. Engineered Meganucleases that Cut within the DHFR or GS Genes.
[0150] A meganuclease called CHO-13/14 (SEQ ID NO: 12) was produced which cuts a recognition sequence in the CHO DHFR gene (SEQ ID NO: 13). The recognition sequence is in an intron between Exon 2 and Exon 3 of CHO DHFR. A meganuclease called CGS-5/6 (SEQ ID NO: 14) was produced which cuts a recognition sequence in the CHO GS gene (SEQ ID NO: 15). Each meganuclease comprises an N-terminal nuclease-localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit.
2. Site-Specific Cleavage of Plasmid DNA by Meganucleases CHO-13/14 and CGS-5/6
[0151] CHO-13/14 and CGS-5/6 were evaluated using a direct-repeat recombination assay as described in Example 1 (
3. Site-Specific Cleavage of the CHO GS Gene by CGS-5/6
[0152] CHO cells were transfected with mRNA encoding CGS-5/6. mRNA was prepared by first producing a PCR template for an in vitro transcription reaction (SEQ ID NO: 22). Each PCR product included a T7 promoter and 609 bp of vector sequence downstream of the meganuclease gene. The PCR product was gel purified to ensure a single template. Capped (m7G) RNA was generated using the RiboMAX T7 kit (Promega Corp., Fitchburg, Wis.) according to the manufacturer's instructions and. Ribo m7G cap analog (Promega Corp., Fitchburg, Wis.) was included in the reaction and 0.5 g of the purified meganuclease PCR product served as the DNA template. Capped RNA was purified using the SV Total RNA Isolation System (Promega Corp., Fitchburg, Wis.) according to the manufacturer's instructions.
[0153] 1.510.sup.6 CHO-K1 cells were nucleofected with 310.sup.12 copies of CGS-5/6 using an Amaxa Nucleofector II device (Lonza Group Ltd., Basel, Switzerland) and the U-23 program according to the manufacturer's instructions. 48 hours post-transfection, genomic DNA was isolated from the cells using a FlexiGene kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The genomic DNA was then subjected to PCR to amplify the CGS-5/6 target site using the primers of SEQ ID NO: 23 and SEQ ID NO: 24. The PCR products were cloned into a pUC-19 plasmid and 94 plasmids harboring PCR products were digested with the BssSI restriction enzyme, which recognized and cuts the sequence 5-CTCGTG-3 found within the CGS-5/6 recognition sequence. 17 plasmids were found to be resistant to BssSI, suggesting that the CGS-5/6 recognition site was mutated. These 17 plasmids were sequenced to confirm the existence of indels or point mutations within the CGS-5/6 recognition sequence (
Example 3
Meganucleases for Targeting Gene Insertion to the CHO GS Locus
[0154] 1. Engineered Meganucleases that Cut Downstream of the CHO GS Gene.
[0155] An engineered meganuclease called CHOX-45/46 (SEQ ID NO: 29) was produced which recognizes a DNA sequence (SEQ ID NO: 30) approximately 7700 base pairs downstream of the CHO GS coding sequence. CHO cells were transfected with mRNA encoding CHOX-45/46 as described in Example 2. 72 hours post transfection, genomic DNA was extracted from the transfected cell pool and the region downstream of the CHO GS gene was PCR amplified using a pair of primers (SEQ ID NO: 31 and SEQ ID NO: 32) flanking the CHOX-45/46 recognition sequence. PCR products were then cloned and 24 cloned products were sequenced. It was found that 14 of the 24 clones PCR products (58.3%) had large mutations in the sequence consistent with meganuclease-induced genome cleavage followed by mutagenic repair by non-homologous end-joining. From these data, we conclude that the CHOX-45/46 meganuclease is able to specifically cleave a DNA site downstream of the CHO GS gene coding sequence and will likely be able to target the insertion of transgenes to this amplifiable locus in the genome.
TABLE-US-00003 SEQUENCELISTING SEQIDNO:1(wild-typeI-CreI,GenbankAccession#P05725) 1 MNTKYNKEFLLYLAGFVDGDGSIIAQIKPNQSYKFKHQLSLTFQVTQKTQRRWFLDKLVD 61 EIGVGYVRDRGSVSDYILSEIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPD 121 KFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKKKSSP SEQIDNO:2(Chromosomalregion5,000-55,000basepairsdownstreamofCHO DHFRgenecodingsequence) 1 taaaactcaagatgccagctttgtagctagcttaggaaacaaagtagtaaaaaataataa 61 tgggtgggtgaaggtctgaagcatttacagagttctctcaagacaaagcacagaggctgg 121 tggccacataacttggcaactgatttgggggaacagaatacaagaaaggaaatttaaata 181 ctgtttttctcaatgttgaactatatgggcatagtcacagctgcctaacctatagagact 241 ggaagctggaacctcggctatctaagatagaataatcaagaaatgtcaattatttgagaa 301 aaacatcaggaataaatagctgctaagttacaagttggtgctttagacatttggagagga 361 taggatgggggctcccagacctggggctccctaataaagctgtgctggcctacaagttcc 421 agggatcctccagtccatgcctcccactgttgggactgcgggcgatggtttctgacgtgg 481 gtactgagggcctgaactgtccacacacttaagccacacgccttttactgagtcatctcc 541 tcatctcagaacattttcctttaatctttcttaatgaaaaggtcgcatttcttccgaggg 601 ctagcctcctgttactctctatacatgtcacataaaactacatgaaaactttgaaggcac 661 tatatgtccatactcagatgaaaagccattagctgtggtcatacaaaaccccacagacca 721 actgttgggaaacatcagacttttttcctgcagcgcctgccctgatcttccacagagaat 781 tcagtctcactttttccaggatgacttctgaactatcaccgtaagatgagaatttgaaac 841 aaagatgtaagtaatgaacttcatgtgttctgaacacacagcttagtgcattgaaattac 901 gtaacacccgcttccttataagccatttctcaaaatgttcccattacacctgcatcgggg 961 atgggtcccagaatcttccttttaaataaacaccccagaggattctgaagctagaacacc 1021 aaggactgacagagagaagcatgcctgtgggcgactccagacacctgggagctgcctgct 1081 ttcttgctactgatttagaaggcatttgcccccgaatggggctgggggactgtcactatt 1141 tctcattctcgggactttgaaaggaagcaaaacagaaaaccatgcaaagtataagccacc 1201 atggaataatggcagacgatccggttgtgcagattagattttacatattgctgattttga 1261 agctaaagacctttcacttcttaaatatataataaaattcatacaagagtattttgtgta 1321 ggtaactcagtcagatacaaggtaagcaaagtaaatgataggtgccccttaacaaaatgc 1381 attctcatagttcatttatcaattatagaaatggtggactggagggaaggcttgaggtca 1441 ggagaatgtgctgctcttccagacagcccgggttcttttccccagcaatctgggactcac 1501 gtctgcctgtagctccaggcccaggggatctggcaccttcttctggcctctgcaggcacc 1561 catacacacatggcatacacacacatacacaaattctaaaattaaatagtaggttgtagg 1621 cctacacaaaaacatgcatacattaactaaataattaatagttaataaataaaaatcaac 1681 caaacacatacactgattaagtaacatgactctgtaaggtcaaaggcggctgaccagctg 1741 tgggaagggttaaataataacaatcacctttgaaagactggacctggtgattaaggatgt 1801 tccagctgtgtcgtggatgagaaatcaaatgcataattgaatgagtgccaggaatagaac 1861 tggagactttctggtgagaatgcttttactggcagtagagtccctgtctaaacaggagag 1921 agacctgcagtagccctgtggcggccctgcagtggccctgtgatggctctgcagttgtac 1981 tcttcctgagataggagacacactagagagtgtttctaatgagcagctcctgtactttct 2041 gttcccctggagaccgcacgtgtttctccgataatacattgacatttctgttaaaccatt 2101 ttcttcttggaacaaaaatggagaacaaatcagattggtgtgtggtcttttaaataactt 2161 ggtacttaataacacaaaacaaaattatcagaggctggattttaggtgctctcagcatct 2221 gccacccctgagccatcagtcaggtcttggaggaacaatctccaaggagaaaacagttct 2281 gtcctcagaaaagctggaggaatatgagattttctacagcactcatagcaaaatcattta 2341 cggaagggatcctgagtaagatggcctcttcttcatcacatggtcatagtctgcttcaat 2401 ggggagaatagttcaatctagcatcgagaaatcgaaggttcccttttgactggcaatgcc 2461 ccatagatagatagatatagattatgtatatattgtgtaaaacacacgtatgtatatata 2521 atacacatacatgtatgtgtatacatacatacatacatacatacatacatacatacatac 2581 atacatagatacgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgt 2641 ttgagactgagtttctctactatgtagctctggctgtcctgaaagttgctaagtagacca 2701 gactggccagaccagatccaccctcctctgcctcctaagtgctgagattaaaggcctgca 2761 cccacccccacccagcccatcttatattttgcttcatttcaaagtaagctctatgcatca 2821 tttattcctgcatattattagccatggttcagtcttgtttgtgttttggaatatttactt 2881 aacaaaacttgaaaaacatttttcaagatttgtttgtttttaagatttatttatttatta 2941 tgtataataataaatattattatgaaaaacggtgttctgcctgcagggcagaagagggca 3001 ccagattgaattacagatggttgtgagccaccatgtggttgctgggacttgaactcagga 3061 cctctggaagagcagccagtacttttaactgctgagccatctccccaggcccaaaataca 3121 catcttaagtgtattgccacaagcatacatcttcatggcccaatcttctgtccatcactt 3181 cagacagctctccttctttccctggccagtcacaacaccctcagctatcaggaaaggccc 3241 tatgggggttgttttgttttcccactccagttcccttgcctgctctgacctcatgagtag 3301 actcatacaggatgtgctcacttcacttgggatgatttctttttcacccattgttgctct 3361 gcccagaatttgttcctttttattgtcttagtgttaatcaactatcaaagccagcaacaa 3421 aaaatagtagggaaacttttttgatagggtaaacctgattgattgcaggctttggttgcc 3481 ttgtttggtctatccccttgagagtcccttacaatgtgagttagttagtggctgctaact 3541 agttgaatctcaacttcctttttctttaatgtgggtatttgtaaggaatagcccccttaa 3601 atctagattctgttctcaaatcaagcaagctcaaggctgtaagcatggattcaccaactt 3661 tcctgctcaaggaatttaaatgtctggtctccatcatattactttaatagtaatagttta 3721 ttatacacatgtgccagctgtatatcccttttcttcttgatggacctatgaactctgttg 3781 aggtgagatttgaaccccttagaaggtgctagagaagaggtacctgatggtcaaggcaag 3841 gctgatacttattcatgggtcccacatctgctaatgtaagcaataacagataatatgctt 3901 tgtgtttagacccacagtggttgcatgtacactaagtatgtatcatcattgtcttatcgt 3961 tcctttagaatacagctaataattatgaccgctattctcatagcatttatattatatgag 4021 cattgtaaattattttgaaatgctttaagatatacttgagaactatgcatatcatgcgta 4081 tgttgttctaccagctgggaccttgaaatgagatcccttgaggccagcataaagagaaag 4141 ttttcatctcaaacaaacaaaagatacacttgataatagatgagggataaatgtcatact 4201 ttttatatagtgattgagaatctacagatttgggtatcctggtcacttaggagaccaagg 4261 gaggactattagctctagagctatgaactttatctccagattccaaagccaatacaaact 4321 ctagccaagttggggtgctgttacctgtatccctctgtcaaattccaagtgttttcacca 4381 cctttactgtatctttccaactgttctcttttataaccacacatagttcatggtctttcc 4441 ttctctcacttgactgtggagtaacctaacttgcgtgtttccagttttcgatctcttcct 4501 taaatctacactagttaaccacaaagaccctcttttctgagctgtgtctattctatcact 4561 gtcaccattccttaatgctctcccagatgcagccaaacttcactttgggcttgagagtct 4621 tctccaggtgacagtgactaatgtctccagattgagcatctaccatctaccctgtgtatt 4681 acacatgaatagccttagcttttcagcaatagacagatagatccatagttagccatgtca 4741 acacccttcttcatgctgttctcacagtaataagtcctaattcctgttttctcccatcta 4801 aactcaaccctgtcctaaataccttactcaaatcctaattgtatctcttccacaaacatt 4861 tcccccttctctccattacaaggtggaaactcagagatccaggtgtcttgcatgttgttg 4921 attctgtcctcaacaaggaattccccaggttcctgcacgaaggaaagcatggaggaccat 4981 acttgaggctactggtgtagtgggaagacaggcccaaaccatgtcacagaaacccatcac 5041 cagaaagttgggggaggcagcccagttgtggagcaggagaaggagaaaacaggcttgggg 5101 aactgctagctatgctttgtcacagtcacaagaaaaaagggccctagcctggcctacata 5161 ttctacaacttcctgaatctttgctctgaaatgaagaggtttggatggctgtctgggaat 5221 tcatcttgcttgcagtgaagctccttggggtatttgaaaccaggaagtttgaaggagttg 5281 atgctaattgttttctaaagtgtgtgaggagtactggcagagttcaggccttgtgaggaa 5341 agaatcctatatctagtctgcactcctgggcacatgagacattcagctatctcccttata 5401 aagcatagaaagtactcttgtacttgacacagaaataatttcagtatgtagagcattaaa 5461 aaaaagtatgaatgacttagagagatggctcatcagttaaaagcacatactgctcttcca 5521 gaggtcctgagttcaattcccaacaaccacaaaaactcacacatatgcatgtgattaaaa 5581 ataaaatctctctctctctctctctctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgag 5641 tgtgtgtgtgtgtgtgtgagtgtgtgagtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg 5701 tgtgtgtgtgtgtgtgtgtgtgtgtgatggtgggcttgtgtttgcaagcccagcactagg 5761 gagttaaggcctcactcacagtgccaggccagtctaggttacagtgagttctagacagcc 5821 caagctacagagtaaggtactgacaaagaaagaaagaaagaaaaaaagaaagaaagaaag 5881 aaagaaagaaagaaagaaagaaagaaagaaagaaagaaagaaagaaaggagagaggtgag 5941 agggagggaaggaactggaagggggaaggagggaaagaaaagaaaaagaaacaaccaaag 6001 gaacaaaccactgtatgccattatacattagctttgggctttacaggttatacactctat 6061 attgtcatagccaatgtctcaatattccataagaggtgtctagttgtgggtatgttcttt 6121 cttagtccttttatttagactacatgacctgtttttgcctaataggccattagtaatact 6181 gacttctccacatgctgccctcaaaacttactcctggaagatctttatttaagctatgaa 6241 cgaaaatcttaaccctgtgacctgccacccagaatgcctctgggaacaacctcaggcaac 6301 ctatcaagccgcttttccaacatttggggcaacagggattaaaattatgattgttgtctg 6361 cctgctgagttcaaactcacagagggaccagaagctgactcactgatatcaagcagttct 6421 aaattttcagtttaaaactctaattattaaacaggggatgtcctcagaccagcactcaag 6481 agaaggagataggcagagctctatgagttgagttataggccagcctggttttcatagtga 6541 gttttagctctccagagagttaccagcaagaccctgtcacaaacaaataaaaacaaacaa 6601 acaattaggggatatacatataactaaatgataaagccttacctagcacattcaagtccc 6661 caggttcaattgctagccctgggtggggatttggacaaatttaaaaagaccttttttgta 6721 tcacacataaatatgactgcactggttgttgttttccatggaaacagaatcaatgtggca 6781 tgtattttacggcattagctcatatagttgtgcaggctggcaagtgtggaatgtataggg 6841 caggccaggaatcagaaattgatacaaaattcaggaaagacctctgggtgcaatggtgca 6901 cacctttaattcaagcacttgaaaggcagaggcaggtgatctttgtgagttccaggccag 6961 cctggtctacatagtgaattccgggacagccagggcttcatagaaagaacctgtctcaaa 7021 acacacaaacaatcagagggaagggcttattttgtttttgagacagggtcttctatgtag 7081 cccaggctggcctcaaactcatgctcttgatatgcccacctcacaagtgcatgttaagat 7141 tacaggtgcctgacacacaccacttttgtgaagtgctgaagagtaagcccagggcttcat 7201 ggacgctgggcaagcactgtgccagctgagccacactccccagtgtgcacgatactttgc 7261 aaagatagatccatatggatgctgtgcttctatctaaacagaatgacaaccacactctgg 7321 caggttctggttcataactgagtcttattggtcacctccttctccatttttcgctggtat 7381 ttctcaaggagagaccacaaatgagaagtgaagcctaacttttaatgcggtctctcctat 7441 gtcacctaaattctagctcaaacagggtttctggctcttaccttttcctcgggtttctgg 7501 atacttgaagtgttaacgggcatttctcttaaagaccaaatctggccagattcaaatggc 7561 tggccttcaactcggcaaactaggaacaataatgtccgctgcatgtggcttgtagcactc 7621 tgtttctattcatggacttgtgagtgatttctgggaaacacgaattataagataagtcct 7681 tttcagtggacttcacaagttcaccctcaggtagtatactgtcaggtagaaacgtctttc 7741 agagaagcgagaggtgacaagccctctgggctggccattgtccctgctggcattgaacag 7801 cctgttcagcacatgaaagcatcgcctgatgctcccaaagctggagcactggcagccccc 7861 tgcagtcaggtgtgtagggtgggttagcaggggtgcttaggcgggttttgtagttacctt 7921 ttcaacacaaatgcaaaagccagagagagagagagagagagagagagagagagagagaga 7981 gagagggagagagagagagagagagagagagagagagagagagagagagagcaggaaagc 8041 atccaggctttgaagcaagccagccttcagctctgtccttgagccattctgagtggaatg 8101 gagtaattgtctgcttggagaactgaagaatagcacatggcaaagaacaatttgtacctg 8161 gaatatattcattagcttgcatgtcaaaaggccacatgcagatagaaaccattatcttgg 8221 cattctttaaaaccttgcagccttgagacttgaggtgcagaaacccacatgcccatgtga 8281 ctgactacctgtcgatctctccagccctgcctggctaacagggacaatatagggggatgg 8341 tgggaggggacagcttagactcctgtggacttggattgaaagaagaacagggaagacagg 8401 ggactgtgcaaataagcactctattaggacctatttttggtgtcttgggaccctcctact 8461 ggtttagcttaaattgagaggggatttggtttgcctcactagctgtttcttcccactcaa 8521 ttcacaattacagctttcttcattgtcattaaaatacattaaatgtgtacttgttggggt 8581 aaggctttctgttgaaatctgcataaagacaatgtccacagcccccagtcagtggaaaga 8641 gcagtaggaccagaaggcatgtgtttccatcccgagtctatattggaatgtttgttaaaa 8701 cctgcacttgtaagagacaaacactagaaccatcagcttgcaggtctacaggccagtgtt 8761 gccagtgcagataatgcccaaactggaacctaaagatgaaggcctttgggagctgaggtg 8821 gaagagtcagctgtgatctcccagatgtcctcctcatgccccattgccactctagcctcc 8881 cacctccaagcacatttgggatccaactgctaacccctggtgttcttttcttagttgaaa 8941 ttctcagggaataacctaagagtctctgtcactcagtctatggcatcctatgataacagc 9001 caaggctaaatagccatcattgttctttttccagatgctcagcaatgaggatgcagaggt 9061 gaacaaaggtggttcagggctgccctgatgatgaatttgacaagccagaatctaacaaga 9121 tcagtcggtaaacagaatcctccttcctatccagagatgttggcttgttctgtcactgga 9181 tgggcatcatttactataagtcatacaggcaccagacactcagagataaataacatgaag 9241 tttccagtcttatgcagtcctgtctagttgacttgccagtattctcaaggaagttccacc 9301 ccagcccctggcatccatagaccaaggactctggaatgttctgggaaagctccacctgag 9361 ctcctagcacccatatatccaaagagtctggaacgttatggtggaagccccacctctctc 9421 tccccagacctcgccccctcaaaaagtccaccaaagactccccaccccccacacaccccc 9481 agatgctcaagaccacttccatagagtatttaaactgcctcccagaaaacagaattcatt 9541 ttttcagtctctcttccccatgtcctctcagggtggggggcaggggtattagtattcaag 9601 cacctatactggcctgtccttggggttctgacaagatatgacctcagctacagccactaa 9661 gatcaccacctgtgtatatccactatgctcccttttaaaagggccctgtccacctcccat 9721 tctctctgtctctctctctgtctctgtctctgtgtgtgtgtgtctctgtctctctctctc 9781 tttctctctctctctgtctctctctctctctccttctctgcctgactctccctccctccc 9841 ctgctctcttctttcctgctgcttttgtccctagaggctagtctcctctctccccttccc 9901 ccttttcccattcactttcccccaataaaaaactctccacccaagctctatcacatggca 9961 tcattctcttgctccatgattttaaaatcacaatgaggaggggagcatggaaaaattatc 10021 caggaagactttatccattaaacctgggtgctttttctttcttccttccttcctttcttt 10081 ccttctttctttcttcctttcttttttcctttcttcctttcttttttccttttttccttt 10141 ctttttgttttgttttgttttgagacagcgtttctctgtagctttggagactgccctgaa 10201 actcaatctgtagagcaggctggccttgagctcacagagatccacctgcctctgcctccc 10261 atgtgcttgaattaaaggtgtgcaccaccactgcctggcttaaaactgggctttttctaa 10321 gtcagtttgatttggattgctgcattggcagagaggtttattggggtgcagaaacctttc 10381 aaccagcttttgagctaatgatagagagaagctcaaggaattggagcaatgcttgactag 10441 ggatgtcagagggaggctatccagaggagcttacaactgaggtaaacttaaaagttaggg 10501 agtttgtcaacttcaacccacagaatagagcagagccaggaggagctgaggcttctgagt 10561 gttatggtggaagcatcaccccaacccttgacatccatatgcctgaagagtctggaatgt 10621 tatggtggaagttccacccaagcctcccttcccggtcgccctccaaaccctgctacatct 10681 cagaaatcccaccaaatgatgactccctcccccagagatattcaagaccactcccacagg 10741 gtatttaaactgccccccaacccccagaaaatagatgtgtggttttccaatctctctttc 10801 ctatcacgtctctggggagctggcaggccatttgggagcattgtatccattaaacgactt 10861 ctcagtggagactctgaaagccagaagagcctagacagatagatgtcttgcatactctag 10921 agactacagatgccggcccagactattatatccagcaaaagtttcaaacaccatacaaag 10981 tcaaatttaaacagtatctatctacaaatccaatattacagaaggtgctagtaggaaaac 11041 tccaaactaagattaactatacctgtgaagacacaggaaataatctcacactggcaaaag 11101 aagaaaaacctctctctctctctcctctctctctctctctctctctctctctctctctct 11161 ctctctctctctctctcacacacacacacacacacacacacacacaccaacaccaatacc 11221 atgaacaacaaaataacaggaattaacaataattgatgtgtgtgtatgtccctgtgtgtg 11281 tgtccttgtgtgtgtctgtttgtgtgtctgtgtatatgtttgtcacctgaggggtggctc 11341 ttccttggtttgtgaggtttctacccaatctataactcccttttcttcattcacttcctc 11401 atgtccttactagtctctattgtggattaaggaaactgtgtggagaacagttttcttcta 11461 gaaaagaacactagccatctcatgtaatcaaattggtgactatcctaattattatgagag 11521 agcttccgtccagtaagtgctagaagtagatgcagagatccacagacaagcactgagcca 11581 agctccaggagtcctgttgaaaagagagaggaaggattgtaggagccaaagagtcaagag 11641 catgacagggaaacccacagagacagctgacctgggcttgtgggtgggagctcatggact 11701 cttgaccaacaattagggaacctgcatgaggccaacctaggaactctgcatgtgtgtgac 11761 agttgtatagcatggtctgtttgtgaggcttctagcagtgggatcagggcctgtccttgg 11821 cgcttgagctggcttttgggaacctgttccgcatgctggattaccacacccagccttgat 11881 gctgggggaagcacttggtcctgcctcaacttgatgcgccttgcattgttggattctcat 11941 gggaggactgcccctttctgaaaaagaacaaggagaagtgaataggggaggggattggga 12001 ggagaggaaggagaggaaactgtgatagggatgtaaaataaattaaaaaattaattaatt 12061 aaaaaagaacacttgtactggtagattggctaaaatgaaacaaagataaaagtacacagg 12121 aaaaagagaggagaaacctggggaggggggctccaaagagaggtgagggggggatgggaa 12181 tggcagcttagtggaggaaggaagacatgacctacacgaatcgagctgtagtttttatct 12241 ggagcatagggtaaagatgtttgaggagaaggaggaacacatgcttgtaaaacatggtct 12301 tcagaaccagcaacaatcatacagagtgtccagggtccatgggcacatgaaggacagacc 12361 aacacatatttaacagtaaagtgtccatatttggtatgaaagtgatgggtaaattgtcct 12421 gggactgtaatttagttgtaaaggacttgtctggcatgtgggtattcttgggttccctcc 12481 ttagcactgaaaaaaaaaaaaaacacacacacacacacacatatattctagtgttttgta 12541 gaaaaggattcaaagaaagccatgatttctcttttgataaatccagaataatgtaataag 12601 aacacacagtggtgtgatttcagcaatcaagtacaggttgcttgtctgtttgttgtatgg 12661 gatggttgggtggttgtttgcttggtttgtaagatgggtgggtgggttggtgggtggttg 12721 cttggttgggtagttggttgggtgattgggtgggtgggtatttggttgggtgggtggtgg 12781 gttggttggtcgtttggttgggtggggtgggttttgttttgagacagggatttactctat 12841 atctcagtttgtctcaaactcactatgtgcacatgagtatgtgatgagattatctaagac 12901 catagtgtctgtgttcatggaatgtctctctagcttagagaatttaaaaaatggccatgt 12961 agggaaacccctcagaaaaggagtttctatggcctccaagaataagaatggatcctccta 13021 gctcggagtcagcaaggaactgaagcccttaattttatagacacaaaggaatccattgtg 13081 tggctccttcccagccaagtctcagatgagtcacagacctgcatggcaccttatgcagtc 13141 ttttgaggtcccaagaataggatgcagataagccatgccagaatcccaacacacaaagcc 13201 ttagtgatatagtaaatatgtattgtgtctaggctgctgcatttctggttatgctactgt 13261 gcagtaatacacaactaatacagatgtgatggttaatattatgtgacaacttgagtgggg 13321 cacagaggtacagacacttggtaaaccattctgggtgcacgtaaggatagttttggatga 13381 cataaacatttagattagtatgctgggtaaaatacattgtccatcccaatgggcatgggc 13441 tttgtccaactagatgacagctggaatagaaaagtctgcctctctcatagttctcaggcc 13501 tttgagctcagactagacagaactcacaggttctctgagctttccagcttgatgaatgtc 13561 catggcagtcttcacacttaacacctgacagacttaatgatcatatgaaccaattcaaat 13621 ctgaccatcactcgggtcattcttttgattctgtcactttggagaactaataccgaggac 13681 ataaaatgccatcacatcgttattttcttcctgtctgtgaatatttttcttttttttctt 13741 ggtttttttttttttttttttttttttttttttgtttttctctgtgtagctttggagcct 13801 atcctggcacttgctctggagaccaggctgaccttgaactctcagagatccgcctgcctc 13861 tgcctcccgagtgctgggattaaaggcgtgtaccaccaacgctcggcctgtctgtgaata 13921 tttaaaatgaaaactttggaaatgttctgaaaccagctggtgtcagatagtcagagaact 13981 ttcgtaaggtaggtgtgggttatagcataatcccacacaagaggctgaagcaggaggatt 14041 ttgtgtttgagggcagctagagccacatggtgagtccctgcctcaaaacacaaaagcaag 14101 acaaaaacaagctccaaataagattcactgggccctttctttccttccttctcagtgagt 14161 ccacttgctttaaaatcaggtcttaaagacgcactagatgctgaacttaacagtaataat 14221 aaatatcttctcttacagtacagattatgctctataaacactgcactgataaagttcagc 14281 cttaacctttgttctgtaaatgtttcctagtttttctactgccgtattataagacaaatg 14341 tcagcatgaaggcaggtttttcagaaaacacagcagctccacagatggcctctaatccat 14401 aatcattaaagacaagactgcaactttttcaactggaaatcattcaagatgtttttctga 14461 agtccctaccaggacacaagccaccctggttgctgtgtgacatcagttaggtagactctg 14521 aactggcttcccaagaaattatacaaaagcaaggtgtcacctagtattagcataacttct 14581 gataactactgtcttagctggggtttctattgctgtgaagagacaccatgaccacagaaa 14641 ctcttataaaggaaagcaattattgggtccagcttacagttcagaggtttaatccattgt 14701 catgattgcaggaagtatggtggcccacaggcagacatggtgctggagaagtagatgaga 14761 gttctatatcagattgacacacttcttccaacaaggccacacctccactcactctgagcc 14821 tatggggccattttcattcaaaccaccaaagctacaaggtagcttataccccagcttgct 14881 atttctgatgagacttagtaaatagtcttaaaagcccataaaatgactcaaaactagttt 14941 ttttattattattattagttcaaattaggaagaagcttgctttacatgtcaatcccttct 15001 ccctctccctcatcaaaactagttttttgttttttaggttttttttcaagacagggtttc 15061 tctgtgtagctttggagcctatcctggcactcgctctggagaccaggctggcctcgaact 15121 cacagagatctgcctgcctttgcctcccgagtgctgggattaaaggcatgcaccaccaac 15181 acctggccaaaattagttttaagtccagttctaggagctccaatgccctcttttggcttc 15241 catgggaaccaggaacactatatatatatatatatatatatatatatatatatatatata 15301 tatatattcaggcaaatatttatgcatataaaaataaaataaatcttttttccttttttt 15361 tttaaagaagtgacattgtcttggaatttttgtggctgctctgcccttatgtgtaactgg 15421 acactaccagcatctaaacactggcctgaaaccagccaaagaaaacctttgtgccaggtc 15481 ctgtgtcaaagtattatgttccttttaggatatcctatatcctaaaggatttattttact 15541 gatagcatcttaacttcctttgaaaggttggtcttctcaagcagtcctcgtggagctggc 15601 tcctcagctaatgccaggggacaataatgatcccctcccaaaaccaaacagaaaaccatg 15661 gcaactctggtttccttgggcagcacctgctttaagaatgagcaaatgaccaatcagctc 15721 atgaaactaaatactctattattactaaaatatttttttgagacagggcatggaattcat 15781 cacatagttcaggttggccttgaactcagagagactcacttacctttgcctcccacgtgc 15841 tggaattaaaggcatgaaccaccacaccaaacataacacttgaattttggaagagtcctt 15901 cttccaatagatttgaggttttgaaaatgtggcacagaaaatatgaattcaaatataatg 15961 aaaacaagagataactttcaactaagtttctataggttcttgctaggaatcctaagcttg 16021 tctgaaactctagagcttctgtttctagcttctgagtgttagtattgtaggtatgtgccc 16081 tgcctcagtgtgatgtttttgataatcttaaagaaatcaaagaaattttataaaagacta 16141 gactgtgctacacaaaaagaatattcagatgccaagaaagagttcttagaaattaagaaa 16201 tatgctactagtataaatcctttataaagtggaatgacaaatctgatgaaatcttactaa 16261 aagtagaaaaacataaacatcaaagacatgaataataagaaaatcatattgtgcatatga 16321 ttaacctaaaacattaacttgcaaaaatagaatagtcccaaaaagtaaacaaaataaata 16381 aatcaccaagaacatgatacaaggacaattcctaggatgataaaacaagaatattcatta 16441 taaaaggccctatcactaaagcacaacagaaacagactcaaaagataaatcttcattgtc 16501 actggagagaagtccatactatcatagcactcagaaggaaataaaaatcaaaatgtcaaa 16561 aaggacctcagcctctgaaacacaaatacaaaatatgtcccgccttcttgacacgcatta 16621 ctcttcaattaacattttaagaaaactataaactgttaaagagagcttagtattttaaga 16681 aatctgtagctatttcttttataagcatgacaactaagtttccctgatttaaacagacct 16741 aaaaaaccggtgaagtgagtggagaaaggggatacgaagacagcatcccacatgactgct 16801 cccagtaaaggcaaggtcttcatccattttatcctgaactctgggaaatttataaagaac 16861 agaaatgtatttctctcagttctggagcctcagtccaggacactaagtctaggtactaca 16921 ctctcacatggtggaaagtagaaagcaagctcacttgtcactcactacctgatgcctctt 16981 tcatcaatcccattgataaggaagagacctggcatctcagtttcctaaggactcagctct 17041 tactaacattagctgtcatttctgggtcactgtaacagaaagcctgacagaagcaaccca 17101 ggggaagaaggatgtattttggctcactgtctctgaggatttcaacttatcccagcaata 17161 aagggataaaggcattgcagcaggaatatgtgtggcagaagctgtttatgtcacaataaa 17221 caaataaacacacgctagcgcgcgcgcacacacacacacacacacacacacacacacaca 17281 cacagagagagagagagagagagagagagagagagagagagagaggggggggggcagaca 17341 gacagacagagagggagagaggcagagagggagagagagagagagagagagagagagaga 17401 gagagagagagagagagagagagagaaatcaaaggcccacctccatcagactggtcccat 17461 atcccaaatttctagaacctcctaaaacaacaccatcaactgagggagacatttttggat 17521 tgaaagcataatgccattacccaggcagaatctgcctgtctgggggagtcacatttaagc 17581 catggtatcaattgacctcatgtaatttcagaatactacataaaactatcagatattttt 17641 catgatgaatttctaaagcttgaaattccctttgaataaaggaccaactacagaattttg 17701 ctgagtctacaattacatacatgaaaatgtaactacgaagtggccagccacaatgaaaat 17761 taaagtgtttgggtggtctgtctctattgatgctcttctttgccctgtttttttttaata 17821 ttgttgatggtttgtttttcttttaagatacttggccccaagaaaaaaaatgacagcctt 17881 aattaattttgtttactctcctgacattttaaaagacaaatttatgaagacctgactgtt 17941 ccatgtagtattagaaagatgtaaaattaagggttgcttaagctgtgtagaattgaagag 18001 cacagcatttgagtgacagggtacaattagagatcatcagggatgtggcacaaagtgtac 18061 tcaacctcaccttttcctgcttagcagagaacagggtgcctcggtgagataggaaattaa 18121 tcaaatagaagaagaaatagtaattttagaaggatcaaattttcctggttagaatgatca 18181 aaactacaagacttgtaactaaaatatagtcaaacccatttcaactggaatctgtgctat 18241 tcatgtatagattaactagaatctaatttttaaattttcatcttacttccaaaaatattt 18301 gtccaaatactctgtgaatgcattagtttcttatgggaaaacatcatatcttttgtacaa 18361 tgtgtttcttagcttgaggttctctccaaacaggaccaagacgaggccaggaccatgtga 18421 tacaacccatagtcctcaagaaatagttgtcattttcttattccaattgcatcccaaggt 18481 ctcatctcattttgcgtgtgcctttgacaccccatacccacataaactaaggtggtgtta 18541 ttttttgaggccctgaaggtatcttcaggaatccataagtgagccttaagctgcatctgg 18601 atataggaatctgaaagtgtcccttctctgcatgatctcttctttcagtttttcaagtca 18661 gtgtgccacaggaatcaggaacgataaatggagaggggaagtgcagttgcttggtataga 18721 caccccagagggctatttgcatcctgtccttcaaaatctctctgagccttcctgcctaag 18781 ctgttttgagttgggtttgtggtaccagaacccctgcccccgccccattctgactaatga 18841 gagagagagagagagagagagagagagagagagagagagagcagcagagcatagaatgaa 18901 agtaggttagaagggcaggtaaaagcactttagacaagagcaggtataagggccttggac 18961 tccctccccagaacacacacatgaaggtaaacgatggttaaaggatacagataggatgtc 19021 gaagctggacgatcacttgcttttgtgtgcttgaagtgacaggctgtggctttcgggttc 19081 atggggtctgttgttgagttcacagtctcaccatgttagcaagcatgtcactattaagct 19141 ctatccccgccccccttttttgagacatggtcttgctaacatacccagaccggcctagga 19201 agcactttgcagtctcagctcccctgagtgctatgatcactcgtgtgagctacagtaccc 19261 aaaccagaatatgtgtgttgggtgttatgagagtttacacattgctgccttgaatgctgc 19321 tctgcttgagttcctgtaggaagctgagctgggaacctaagcttcctcctcccagatagc 19381 agtaaccctgcagagacctcccaccaagactagctaacccctccttcttgtgctgtactt 19441 agcaagaaccccaaggttctgggtccttgtgctacagttccagaagagtatgaacaatct 19501 tagcttttctgtatatgtgtctgtgtctgtcctgtcagatcaagtcccagcctcactgta 19561 tgcaacatgaaaggctgtgaaaactgtgcattttgagaatgaacatcattagtctccagt 19621 aagttcaaaaacaaatgaaggcagccactcataagggtctttaatgaggcaagggggcaa 19681 aagggtggtttctgtttgttcaaagaagcctgtcatacattttcagaaaatttagaaaca 19741 cgtatcatgtcatttcacgttagtatgaagtccttataattcatttcatattaaatgatt 19801 tcctttggttagaagcaaaattatgcataaaatgtgttcctttgtgtttggagcaaaatt 19861 acaagttacattattagttaatattctagttcttatttttcccaatctccaagaagcaaa 19921 atattcccctaaaccctaaagcatcaaattatcctatcacacagtgaccagtcatcgtaa 19981 cctaaatattaaagcatcagattatcctgtctatggtgaccagtcattgtaacctaaata 20041 ttattgtaatgtggattagagttaactataccttttcatcacactataatgtaaacactc 20101 tccaaatctttcaaagtcttgaaaacacaatttataaatactgtgttctgtttgttttga 20161 gacctgatccggttaggaatttcaggctgtcctcaaactcatcatcttcctgcctcactc 20221 aggtcctaagtgctgagattaaaggtctatgctaccacagccatacgaatgccatgtctc 20281 catcagcttatcacttcttaacttttttcttttcttcttctacatactgctgagtaggag 20341 catcgatgacctcagcctagtaggaatggttcccatgtgaacccttaatctgtaggaaga 20401 tgctggacttcttccattaagactgatctccatttgaacttgacttgtctctctcttgtg 20461 tggagctaccatcccatatataatcttctggtttataaacagattgctttaccctcaaga 20521 tcctttgctagcgcagcaatgtaagttttaatacaaacagtaaggtctctgattggagtg 20581 tcatggtttggttaagtgccctttccaagggcccatatagttaagggctcaaccaccaag 20641 tgatgcttgtggataggaggcagggcctagtggacagtctttaggtcatggagctatgct 20701 gttgagggggactgtggggtcctggtctttttcccactcctttttaggtcctagctatga 20761 ggtgagtggttttgtcctatcaagcacctctgtcctgccatggtgtaattgattataact 20821 acaacctctgaaactaagccagtataacctatttatctcaagatgtaacttacaggtaat 20881 ggtaagataaagctaacaaaagacaaattgttataatccaggcaagcctggccccatccc 20941 ttgggggcatggcacagagtgtgtcacccatctgtgcatggcaagcagtaccctgactct 21001 gtatgctgattcaaaggtcccttaaagcaaactcctcccacttcctctctttttctgcca 21061 tttctctgaggagggaggccactgtctctctgtctctctctgtgtctctttttctatctt 21121 cctctccctctcttccctttccccaataaactttccacattaagttttgtctgaaggtat 21181 ctgtttgtctctcacccgccttttaggccccacctaccatgggatctgccaaaggtctca 21241 cctcgagctgtattcataacacaaatgacagacaaagatcaaccctgaagactagtagga 21301 tgtagaaggcctggagctgacctgaagaacactgctgacttcaacattgcccatccgtca 21361 gttatgtagcattaaagttatagtggttcctcagaaagcagtctcctttgaaaacttctc 21421 gttttgtgtctaaatggaattaaataccttgttcccgaataattgttttagttctcttga 21481 aagatcccgtatacttactattaagatgtatataaacctcaagctgaaagaatgacttcc 21541 cctatggccagatcacaagactctccactgatgtgcccgttgcaacctgattagaggaag 21601 agggtcaaagttccccaagattcagctgagttcatgcaagttttagaaaaaaaacaagat 21661 gttcctccacagttagaaaggagtggggctggagggatgactcactgagaaaggttattg 21721 tcgtacaagcatgaagacctgagctcgaagcctggcacccatgtaaaaagaaaccatgca 21781 tggtagtgtgcatcttcaatcccagcattggggagacagagaaagagaaagggacatccc 21841 tagagcttcctggtcagccagccttggcaagccagtgaactccaggttcagtgagagacc 21901 tgtctggggaggaaaaagggagggagggagggagagagagagagacacacacacacacac 21961 acacacacagagagagagagagagagagagagagagagagagagagagagagagattgag 22021 gaagatacctgatatcaacctcacacactcatgtacccatgtatgtaggtaccttcacac 22081 acacacacacacacacacacacacacacacacacacacacacacacacacacacacacac 22141 acggatggtgttgaattctaaggctcttatccacacatatatggagacaaatagaagaat 22201 tacagtcgtccctgcctttgacgctactctgtttctccaaccctgcttcccagatatttt 22261 tcaacatctactcagccttgagtggttgcactctgaccccaggacctctttctgtgactt 22321 ccttggcctcctgttttgtttttctgatgctaaaaactgaatctggggcctcatgcacac 22381 aggaagatgctataccaatgagctacaattttgttgccctttttaatttttgagatggtc 22441 tcactaaattgttcaggatggcccacttgtaattctcctgccttagcttcccaagtagct 22501 gggcttttatacagatctgtgcttccacacctggctgagcagacactcatgatttcattt 22561 ctgctaatcaggtagttttcttgcccctcgctgccatttcctacctgcctttccttgcca 22621 actaaactggttcccacaagcgacaggctatcatttctcagctcttccacaggttagctg 22681 tgcaatttggtatgaatcatttagcaagcccagttctcctctttgtaaaacagatgattt 22741 agatgaaattttttcaaagttctctttgaattaaaactatcactgccttgcttgctctct 22801 gactcttggagaccatggcctatccctgattagtccttggtccacagaaggatgggtggc 22861 attggatgtgctgaacaatcaggtactttcatgtcacttggagtcttacagtaactgcat 22921 gtttcaaatgaatcctttctggctctattagtttcttttttgtcactgtgaaaaaaacac 22981 ctgaaagaaacaaggcacggtttgttctgactctcggttcagaggatatagttcaccatg 23041 gaggcaggagcttctcacagctgtaacagccatggagtcaggtggctagttacagtcagc 23101 tggccttagcagtcagagagccaagagagctcagttgaggagagtccagccaggctgtag 23161 cccttaggacctgctccccagagatccactttctacagtatcttctaaacagtgtcacta 23221 gatggtgaccaggtagtcaagcacatgagcctgagggataatatcattcaaaccatagga 23281 ttagtctagaactgaaccagatcaagaaccaggttttcttctcacataatagataccaca 23341 catcatgttctcatatagagtgtgatctaggtattgtttctccaaatggagaagccaaca 23401 ctggatgacttacatagaaagaaagagagggaggaaacaagcaagggagggggaagagtg 23461 agaattattggaacagtaccagtgcctcaaaatccttggtggactagagaattagcctca 23521 ggaagaagcgactaggcttcttacagcatagacatacagttcttaccagaggcacagcca 23581 tcatgggtgccatggggagcatgaagttcagctccatccagccattcctagcgatttctg 23641 gcaacctctgtcctttgagacacttcctgaagatataagagtccagggagagacatctga 23701 ttgctttgatcccaggatcttgggatggaattggtgttgtctctgctccagctccagggt 23761 caggaaggtgaaactggaaacacaagctagcttttcttacttagcaaaaacccacaggtg 23821 acataaaagacagattgacacgagaacagcatggcagatttatttagtcaaagttttacc 23881 agacacaagcaccttcagaaaggtaaagtcagagaccttaggggaattttcttgccagaa 23941 tttttccagaagaatcaacagccgtgtaacaataggactagataaacaagtaagactgga 24001 cctgcagcacaaatgtgacaataggagttggaatccccaggactcacataaagccatggg 24061 agccgaatgtaatggtcacttgtagtttcagcctcagatgggggtggggattctccagaa 24121 taagcaggctagcaagactagccatgttgccaagctctgggttatattgagacactctgc 24181 ctcaatgagtaagtggaagaatgatggaggccaacttcaaccttggacttccacatgaac 24241 acacatacacaatgcaaccatgcatccacagtgtatgtacacacacacacacacacacac 24301 acacacacacacacacacacacgcaaatggacaaagaaagaggtaaaacctacaaggaat 24361 caactgaacagaagccaactggtctgcctgttcagatcctttttggcctctctgtgtgct 24421 tccctttctcctgggcatggggcaggcaggatctgtatggggtgagggtcttcagagaag 24481 cgaacagccttcctaggttttatggctcagtttggtggagaggggatctagtttctctta 24541 atcatctttttaaaaatttattaatttattttttatattccaatcccagttttccctccc 24601 tcctctcttcccctcccccacctcccatctgttccttagagagggtaagacctcctctag 24661 gaagtctactaagtctgccccatcatctcattgaggcaggaccaaggcacctctccaccc 24721 ctacactctggtgtctaggcagaacaaggtatctctccatatagaatgggctccactaag 24781 tcagtttgtgcattagtgttagatcttggacccacttccagtggcctcatatattgtccc 24841 agtcacatcgttgtcacctatattaagggagtctagttcggtcttatgcaggttccccat 24901 ttgtcagactggagtcagtgatctctcactagctctggtcagctgattctgtggtttccc 24961 catcatgatcttgactcctttgttcatattgtcactcttgcctcacttcaattgtactcc 25021 aggagcttgcccattggttagttgtggatttctgcatctgcttccatctatttctggaag 25081 agggttctatcttctctggggttgtgaattgtagactgggtatcttttgctttatgtctg 25141 gtatatgcttatgagtgagtacatacaacatttgtccttctgggtctgggttaccccact 25201 caggatgttttttttctagttctgtccatttgcctgcaaattttagaatgtcattgtttc 25261 ttactgctgagtagtactgcattgtgtaaatgtaccacattttctttatccattcttcag 25321 ttgaggggcatctaggttgtttccaagttctggttattacaaataatgttcctatgaata 25381 tagttgagcaaatgtccttgtggtatgaatgtgcctcctttgggtatatgcacaaaagtg 25441 atatttcagggtcttgaggtaggttgattcctaattttctgagaaatcgacatactaatt 25501 tccatggaggctgtacaagtttgcactcccaccagcaatggaggagtgttctctttactc 25561 cacatcctctccaccataagctgtcatcagtgtttttgatcttagcctttctgatcagct 25621 taaaatggtatctcagggttgttttgttaatcatcttgagaaaaaggaattctattttct 25681 gtgactggctctgagagagagagaagagggaaaggtgggaggaatgtgtgctttcaagac 25741 cttgtgttctcccttagctcaaagtactcaccatgaaaaaccaccagcctttggaggagc 25801 atgctcttgcagaggcaagatcctggcttcctcccatcttgaatttgccaaaatagcaaa 25861 gatgtttgggtgctggacagccaaaaatgacagctgctcacttcacagcttcctcacgta 25921 tgattacaactccactcatcatcaagctttaattacatcatgagcaggcttatggctgag 25981 ccgttatcctcgcatcccttcgtctcatcactgattcacacaaatcactaggtgctccgg 26041 ttaatgaaaacatattcatcagtacagtgactaattcatcaggccaacatttacatggct 26101 cctctgcatgacaaaaatgaatgtttagaatgaataatgagtcaccagaggtgggggaca 26161 tcttctgagcacaggttgcccttgtctttcctggtactcaatcccggctgaagagctgaa 26221 caaagctgaggttatttttcccatgacagtgcattgtggtttagagatctgtaagcggct 26281 tatcttgattggcagtttgattggttctgggatgtactaagagacgtgcctcatgggcat 26341 ttccagaaagaattaactgagggggaagctcctcgccccgagaatgggtaggagcatctg 26401 gtggggtacagatgtaaagtggtccaagggagaagccgcatggcctgcctgccttcactc 26461 cttgctgctgagtgtgtttatcccatctatcccgttgttgcttctgttgcagttgcaatc 26521 ctgcttctccaggccccagcgtagactgaacagtggctgcccagaaattcccaattgaag 26581 cagccgaatggtggactgagcacctctcagtcttcagtctctctagtttgtaggcaacca 26641 ttgttggacccaactcttagtagtaagccaatctactaaatacagaaaggccagtgagat 26701 ggctcagtataggtgcttaccaccaagcttggtgacccgagttcaatccccaagactcat 26761 aaggaaagaactaactaccgagagttgttctctgagctccacacatgctgaaacatgggc 26821 ctccacatgtcatgaacatgttcacacaatacatatttatctctatatattcatttctta 26881 taatttttagaaaatttcattttatgtatatgagtgttttatctgtttgtatgtctgtgt 26941 accacatgcatgcctggtgcctgaagaagtcataagaacgtatcagattccctctaactg 27001 gagctaaaagaagattgagaggtacctaccatctgagtgctaggaaccaaacctgtgtct 27061 tctggaagatcagtaagcatgcttaaccactgagccatcatgccacttatttgtaacaca 27121 tatccatcctattggttacagtcctgactcatacagttagatagctgaggaacctagaat 27181 tcttctgcttttttattacaaaacaaagaattttatctgacttacagttctggccttagt 27241 cagggagctgcattgggagatggcttctctactgtcagagtccagaggtggccgtaaagt 27301 atcatatgacatgaggcagaaagtctaacttacttgagagttaacttggaaatgtccaaa 27361 gagacagggggctaagtccctcttattgaagagaccttccatagaagttagcctgacaga 27421 tggccttgcctgaactgcattgacagtcttacttggaaggcctgttttggttcctaagaa 27481 attcaaggatccaccagagaagtgtgcagccagcaagctggactccctatcccaagcccc 27541 agctcctcctcagggacctcagcagtcctgtgtctagcttacctcagcgatggggggaaa 27601 gatgctgttttcctgctaagagcacactattttatattattgttgacacaggttggactg 27661 catgtaacagactctccaacaacacagtgaagatacaagtgtgttttgctgcatttaaat 27721 gtctccccatctgtccctgctaagacacctactgtccttcacatgtcactgaaaactcca 27781 ccccttatgagaagtcttccctgatgccatctagacaagctaagagtgctctgctctgca 27841 ctgagcagcttctcaactctggggttatcattgctctgcatcacaattagcacacgtggt 27901 agtggctgtgtttgtgtttttccacaccatgagtccagacagcatccctctcaccagcac 27961 gccataggcacaagtgctcaagagtagcaggacttgaacatgtgtggtttatcatacaga 28021 cagctgctgctcagagaccagatcaaattcaaagcaaaatagagagatgatggttcctgc 28081 catgagcgtactgaacaaggacaaacatcaccatcataaggaactcagctgacagggagc 28141 ggtcaccaaacttttttttctgtaaagtgacaaaaatagttaagtattttgccctagaca 28201 tagtgggtggtacacatgtaatctcagcatttgtcagagtgaggcagagagttgaatgct 28261 gggctacgtagatagtctcaaaaaataaataaataagtaaataaataaataaataaataa 28321 aaggaagaaataaaaaaaagaatttgttactcaactctgcacaatggtgcaaaagaaaca 28381 ataagcattatgtaacctagtgggtattggctgtttcactttactaacaggcattgaaat 28441 ttcaattttgcaaaattttcatgttccatattacccttatttttattctcccctataaat 28501 ggtgactcaccaatacgcaactggataagattagggtatttttattagggaatatgcctt 28561 acttacagagcacctaaccagccagcaggaaacatagtaaagtagcgcatgccgatgaaa 28621 caaggaaaaagaagaactaccatgtgtgacccctaacccttaaaacctctcccacatcac 28681 cctgaccatgcccattaggcgtggtcacctagccagcccctaggaggcatggttacggtg 28741 tccccctacactcccctaatcatttaaagatgcaaatgcatgcttggtgatgggctaacc 28801 ttggctcatgggctaatcttggctcatgggctaaccttagctcatgggctaataatcaag 28861 gtttactaatctctgtcagacagccattttttttttgcagagaagaatccccatctttgg 28921 atcatttatttattccttttgtatatttgatgcaatttataaccacaagaacctactatg 28981 tgactgcactgtgccagatggcagagaaagctaagccccgattcttgtggcatggactca 29041 cacaactccagtacaggactgttagtgacaatctccttaaggcataagcatactgcagtg 29101 gcagcctctgggttaggagacaaggatacagtttatgacacctggtatctggaaggcatg 29161 aaacatgtcaaatgctggctacacctaagaatcagcaacatctagtctggccatagccta 29221 ggatgaatgtcacagggtcttaggccagaaatgtatggccgagctgtagcagggtcctct 29281 ctagggccagaattaattccagtgtgatggacagccaagaccacagggataacaaatgag 29341 cagtgccaatgacacgtgcttctccttattattgctgcacagtgtttgttacacatagca 29401 ttttcgcacagtaatataatgtgcttgggtcatcttgcttcatatcccatcactccctcc 29461 atctccctagtgcctcccctgttacctttgcttctcagttttgtttctgctttgatgtca 29521 acagcacatacaagattttatgcaatacatcacttcctgaatggctctatttggaaatca 29581 ctaaaaggtaatttatggaacatttggggtctttttgattttctaatttaccaaaaaatc 29641 cacctggggaaagacaatggagttcaaggacttctaagaggggaatgtaccatggtatgc 29701 tccagccaggggaaccagtgcttcccaggagctatggcttacaaagtgggttatcacatg 29761 aaagcaagactaaaataatcatctcaaatattcattagatgtgggactcctaaccatctc 29821 acaatgcctccctcggtctacattaaataagaaacctccattttgtgctttgcgagaaaa 29881 tgactgaagattatacatttggccttgaagtggaagtatttttgaaaatcatgaatagga 29941 aaataataaatctctcatttcaacataaaatataagggacaaggacatctactcatgctc 30001 caaggacggacactgaattttccatcaggtagttgcagaacgctgtgtcgctcaatcaaa 30061 aattcaggatgcattgctcagagtgcattatattaaaagatagcatcttggaacacagga 30121 tgctcaggaaatgggagggacattaatctgcatgcagtgatcatctcctgcaaagcgggc 30181 atgagagcctgatgggagacaagccatccagatgcccatacccaggggagctgtactggg 30241 ctgcagccctgcgccattcagccatgcaccaggctactccctcctcttccagctttctcc 30301 ttctgatggccataggattagaagataagggactctagtgcaggtcaactgctgaccagt 30361 gtgaaaatgcacagactacatgctggtagatcagcacttcaaactactgttcaccatcat 30421 ctctggaataagcactacatttacagggttcaaacctcaatgaatataaacaaacaaaac 30481 acacctcccttccttcactgtctcccatttctttggttcccatctccacatagaatttat 30541 aattaaaatttctaagtatctttccagaaatacttcacacatgttataagcaaatgtgct 30601 tttaaagatactattttaaattatgaaaatggttatattagttgagataaaagaatagaa 30661 tgggaagttccagaatttaaggcctcatatgaaaatataaagcgctttctcttttaagtc 30721 tagggtaggtgtactagatcagcgctcagctccataccatgaagccatccaggagtcaga 30781 cctctctgacagccctgccattgtcacagagaagtttctgtcaccagtgctcatgctgtc 30841 agaggagcgaaggagaaaagatgtgagacctcccaagtcaaagtcatctatggataaaac 30901 cttagttgcatggcacaccagtgttagggagtcggggaaacacagccatagcccagcttc 30961 ctctctgttcttgctcttattaccaccagaaagaggttgcttagacaacccaaaccaaga 31021 cacagggctctgtgggagggaatcagtcccaggcttctggcacatgctatgtcaccggaa 31081 agccccagccctactccgaatccccacaagtacagcaaatatcagattatagcatttaaa 31141 ggggcactcttgccaaagagaagcaccattggaatagccatgcttgagaactggtcctac 31201 ttactgcagaaccatggatacaggctcccttttgtagatgggcttaataaatacttctat 31261 aagtgatactctgctttgtgaaaatgacctcgtcaatattcaaagtaatcctctggttta 31321 ggactactatgaacctgtggggttcattgttcatgtggttaaacagcaaagagtagttag 31381 acagttgtcctacgtcacagagggggacatatgctatgcttggttaaatagctgtcctgg 31441 tcagaggggaggcatgctattctgccctttctgacagaccctgattgcatagacatttca 31501 gtgagataaaggaaggaagggaagaaggaggaaagacaacattttttgcttctgttaagg 31561 tagagactatctgtgatccagttcagcacagtgcctgtgagtagaagctacaggtcaggc 31621 aggagccaaggaaatgtattgcttttctaattgaacaaaggacacacagctgccatttat 31681 tttcttcattttgacccttcagccctgcactgtggatatgacatcaagaaactaagcagc 31741 cattttgtgaaaatgagatctaagttagtaaatgtggctgaaaaagaagccagctgcatc 31801 ctccctggatttacgagggggaaatgtaggcatactaaattaaaacactaaaattgaccc 31861 aaagctattttgactgatatttaaatatagattctgctcctggacattccagagttcata 31921 ggacagttgcttctgttcagaggattcctcttcggggttgcctctccttccttaggcctg 31981 cttgtcctgcccaaagctgcccaagtgcatcaggccccaaaccaacttctccatcctgac 32041 gcacagcagactaaatatgcaactttgtgtctcttcatcccaggacaaaactttcaccca 32101 gcccctgacatctgagactctactacaggttatctattaaatcttttataaagaccaaga 32161 aacaaagtgttggcatccaaactttggtaaatcatagccttttaataaagtcaaatggac 32221 caatgtactctaacaaaaaaatatgggtctctcatttctgaatggcagatttcaagccct 32281 aagaaccacaatgctcacctactgggcaacactgagttacagagacccagctcccccacc 32341 cctcaccaagccagagaaacactctatctgaacaatccttggtccatggagcaagaatta 32401 gacatagaatttgtatctcattgttttttaggaaaaccccaaaggctattatgaagtcag 32461 tttttctgggcaccttttctttcccatgacaacgagttgtgggcagtctcagcagaatac 32521 tgaagctgtggcttggggagacagagcatatactggattggagttcatgggtgggtgcat 32581 ggaatcaatgccgggcatgggattcaagaccttatgcatgtgggtagatgctttgttact 32641 gggataaatcccccacctgggatctgacttcaagcacaatctttggaaggcggcattggc 32701 tctctgctaatttttctagcacttttattccacttattttctgcttgtttgctttgggag 32761 ttttgttcgttataagacagtcttgctgtgtatcctaggctgatcacaaacctgtggcag 32821 tccttttgtcagcaggccaaaattcccactttatctctgaagacagaaagtagattgagg 32881 aatatatgataaagacactcatcaaagccaggcatctatctttacttttcttaaagcatg 32941 tttttgaatggcataaaaccatgtagacaaggagtcttatgttgtacatggtcctacttt 33001 gtcacttacaatataggatactttcaataagcttggtagcccttgccctattctacttat 33061 tctgttctctcttcctcgggtcttggggagccttcttaccaggtggggtggcataaaggg 33121 aaaagtcacaaagctcttcctattcctggttcccctcctaagtgtaccttgctggtggcc 33181 ttgctagcaaatgtagtataacatctgacttatctcctctcagatatggttgttgtactt 33241 agataaatttaatctagaaactcaagctgtatgtctttggggaccagcattacagagctc 33301 ttcccttcctgtccttacctcaccttggctactgtagtaagttaatcctgatgattcctc 33361 catgagtcctgaaactgattagttccaagagctggaggatgagaagggatatagcctggt 33421 gcagggacactttccaatgaccacaagaccttgcacaaggtacacatggaatgtgttaga 33481 ctgtctcctttctgtccctagcctcagttgccccagtgtttatcaatgtttattaacatt 33541 gccctagcaaaaatactacagactaggaagcttgggtacaattgaaaagagcttctcagg 33601 gttctggataccgggaagtgcaaaggttcagcatctggacagggctgctattgtagtttc 33661 aaatggttctgctgcaacacccctttgagagaatgaacactgcttttcacatggtggaga 33721 gtgcacagacaccaacccaactcctgaaggccctttctcgagggctctaatccatcatga 33781 gggccatactctcaggactcattacctccccaacatcccctctctaaatagtaccacact 33841 gcatttgcatttcaatatatcactggagatatataaatctccagaccacagcataccata 33901 aatcagataaggcaggcctgccttctatagcctttcactcagcaaaggtgtttctagccc 33961 aaagcagtctggactctcactctgaaacctcttgggagtggtggccagaaatgacttccc 34021 atcatccctctctcctgacctggtccagcaccaggtcaccaggaaatcctccaagtttca 34081 ttatccccacccccaattgtctcttgtctctagcaaacctcttccaatacttccttcctt 34141 ggtgggtgtagcaagccagatgatagcctgccaaagaagttcacagcctcatttctggag 34201 cctatgaatatgttacattgtgtggtaaaaggaactttgtaggtgtgattaaattatgaa 34261 tcttgaagtgggcagattatccaagtgagtccagtgaaattgcaaaggtacatcaccaac 34321 agtgaggcaggaaggccagagggggagaaggaagcagagaggcagagggaggaaaagaca 34381 agccaggggaggggagtggggggaaagaaaggagagagagagagagagagagagagagag 34441 agagagagagagagagagagaaatatcacacacacacacacacacacacacacacacaca 34501 cacacacacacacacctgaacctgattgtggaggaagaaaccactaaccaaggcattcga 34561 ggcagcctttgaaagtcacaagagacagggaaaacagattctctccctcggcccttcaga 34621 atcaacacagccccacaactgctgattttagtcatgttaaagccaagttggacttctgac 34681 tgccaaaactttagacgagcaaataaatctgcactattttaagataccaatgtgatttgt 34741 tcatgaaaacaatcaataaggaactaataaagtagaagtgaaaattggatcacttctgaa 34801 gtttggtaatatccacagaaactggacacatgctgactttgtgagccatagctccacacc 34861 caggtatgccccctacagaaatgtgtatataggtgggcaggagatgtcacctgctgtgtt 34921 catagtcgcacctttagactttcccaagcctgagaatagcccaaacacctaccaggagca 34981 aaataaattgagatatacagacgcagtgggatactacacttctaaaagaatgagaaaacc 35041 acgctatacactgtatatcgtcggaacagtaacacaggggtgacaatcaggcaataggac 35101 atattctctatggctttagaaaacataaaaatagcataacagttctgttagtggcaatgt 35161 gttctgttttgtgatctgtatgatgcttcggtttgtgcaaaagctctggacttacctttt 35221 aaatgtatggtggtctataccttttaaatgtatgctagatatacatgagtaaaaatgatt 35281 aaaagagatggaggggaggagactcatgccttcataaaagtttgttctgtcctttctggc 35341 actgtccaagtgaatgtgtgtaaacaaagagtgacccaccccaggtagtccaccttctta 35401 gaacctacttctgctacaacatgtcctgtgaatgtgcaccaaatgtttactaagggatca 35461 tgccacagggttttgtttaaataaagtatgtctacctaggggtatattgattgtctttcc 35521 ttttgagggggggtctcaaaactacaaactagtttgttttgagacaagtatgtagcccag 35581 gatggccttgaactcacaccttctgtcctgcctctttcccagcactaggatggcaggtga 35641 gactatcagcctggccccaggaaactatctttgattgacattatctggtcagaaaagatc 35701 taccttttcctccaccaggtcctccaaatacatgaagagctgaaacagttctgtctaccg 35761 aatttccttttttcttgatgtttctgtggaatttaatacataaattttaatttgcatttt 35821 tagcttttctattaagccttaattagagtataatgaagttatgaatttataaaaataaaa 35881 acaaaacggttgctcccacaatcactcagtcttgaagtgaggttctgactttacctgaag 35941 tgggggaagagagtgaggaaagggacctgcggaagctgaatctcagacccacaagatgga 36001 tctgagatccatccaagcgaacgtggacgcagacccggagtagggacatccaggggtcat 36061 cttcatctgtcctcgctgtgcttctgcccctttgctcctctaccagtctcagctgtcaaa 36121 gctcagtggcctggaggggagatggggcggggcttaggatcgaaggcggagcctcggaga 36181 gcatcttctggcccccggggcctggactggcccgccgcccccacctgcagcgcggcggag 36241 cgcgggcgcgtcactcccagcggaagcgccagcctcgcgtctggcgaggtgcgcgcttcg 36301 cggctcccgctccagagcttcgtggcccgcctgtgtctgcagagcaggggcgggggcccg 36361 gcggcaccgactgggcactgagatccaagtagccactgaatcgtagacagtcacccagct 36421 cggacagcgcgtcggggcgggagcagatcgggaaggtgaaggaccactgcggatccgaca 36481 gcgcgtcccaggtcagtcctcccgctgcacttggggaaactttgggatgcggtgacggct 36541 gcgagatgaggacactgagggtcgcgaggccgcgtggcccctgtgaaccccgcgaacccg 36601 tacctgccgcgcacctgacaccgcagctgccagggcggggaccgaNaccctgctgccgcg 36661 gaccactgcgggccaccaagggctagcgggcttcaggggcctctcgggagcctccggctt 36721 gcccgcgcccagccgcgcgcctccggtcctcgcgggtccccagctccttttggcggctcg 36781 cgcccggaccccgcggggctgcggattccgccgtcttcgggcctcgtggcgctggaggag 36841 cggcccgggggcccatggctgcagggtggcggccccgcggcgggagcggcgcgtgctcgg 36901 ccggtggagcgcgcgggtcgcggggttcggctggagcgcgtggccgcaggtgcctgtggc 36961 cgctgggcagcggaggtgagagcgcgggctggggacgcggagcggattgcaacctctggc 37021 tgcaggaaccagggtcgctgggtgagcagtcctgtccccgcggcttccgggcgtgcacat 37081 ccctggcacccggcatccagaccccatcagctggaggcgggctgcagagcggcgcctgcc 37141 cgggccgaggaccagtgcctcctgctctgacacgccatctcaccaacgagggcggggtgc 37201 tagattggcgggctgcgcggggaccactggccagggccttctggcacaagcccttttcgt 37261 ggacagctgcctgctctggcttggagtggaggagacgaaatgagtaccccgcccccatca 37321 gcgccccaacactgtcgccccagtcaccttcctttgcccttctccgacagcaccttggac 37381 ttgctccctcccgaattggggaaaatctgaggaaaccaggcagggaccttggagataccg 37441 cagcctgcatactcaacagcctggaaatccagtcaccttggtacctcgctgcttcccaga 37501 cactttggaggagcaggtttgccatttctaccccacatccgtaccccatcccccgtccgt 37561 ctctgctgaggaagggactcttatgagagaagttgggatctaggtaccccttaaggtagc 37621 cccagagtctgtggtaactaggctcataggtaactaaaaggcatcctagctctgtagctt 37681 tgtgagggaaacaaaccttaccaactaattccttccctttctgaatatttcttagaagac 37741 tggagaccaacggaagccgactgttctggccagtctttgcaccctttgcttggctctgac 37801 tctccttcctaggcagagaaacattttgcttatgacctctggctggcctccttccaatcg 37861 ctgcctggccttggactgcccatcaggactgtgatttttttttttttttaagacctgatt 37921 aggaaaggctgcaagcctccggttctagaaggctcaaactcaggggtatactcttctctg 37981 atacccatgtgctccctaattccactgtggcaacacctctgcccttcactcccacaagaa 38041 aattggttgtcaaacctcttggggaagatgatggaggcatccctgtgggagcagatgcag 38101 gatttggaagcaaccaggaaacaaccaggagtgaggaatcttttttaaaggctcacatga 38161 ttctggaactaagaaaagatggagatgccaccagtgtatgaagcttggcctctcctcggc 38221 ccatcccacccaactcagggaactggcatatgcaggacctgtattgggtgatgcatattt 38281 ggaacctagtacttattgaattcctaagcagtaaacacattccgaatttgaaattcctca 38341 caatcatctactgNaatgtagatattaaacccccaacttatgaatgatagccccaaaatt 38401 gttaacattgagagagcccaggttccctgccacctcttccacaacaggacaggaactagg 38461 acaatgaataggaccatttgagctttagggtcatgtgcccactttacagctccatagcca 38521 gacaactgttttataagagagggcacaaaggaaaatcactgtcctgtccaaatgaataga 38581 aagctggggatggtggcaggacaaaggcaacaggaaaaatcatctccaacaaggctttcc 38641 aagcatatcagtcttatactactgccatgttgggtaccacacaaatcaggtatctcaaac 38701 tggacgctgcctagggaggtctgtcatctaaaaaggcagggagatattgagataaaatac 38761 acagaagctagtatttaactccaggctggcagataataggaatgaccttgggagggtgtg 38821 cttacctttccttctctcttgaacaaaatgtggactggaccagatgagcaccaaggctcc 38881 accaactctaacagaccttgtgtggtgggcttgcctgcaaacagacttgagctaggttgc 38941 tgtgcgtgggatccattccagactcatttacaaactcgtagtcagtgaaatgtgataaac 39001 cgaacactgtagggatttctaaacaaggaattaaaaaactcgactccaaatgggagagat 39061 gcaggcaacaaatcgacagtgtttatgtgcctctgaatagctttgatttccttcggtagg 39121 agctgacagctggctgacagaaagctcacccagggagagaagagagaaaaatcaagtatg 39181 agattaggaataatgttttcaggtaactttctattcccattcggagtgggtgtctggaag 39241 ggcgagtgtagttatggcttgaattgctccatttatccacagatattttcttcccaaggg 39301 ctcctgattctaagatgctgggctttgcttctgtctcctagtttcctggtagcagggtag 39361 agagctgggggtcccagcattcagcctgcatattcttcctctatcctcactatctgctgc 39421 ctccattatttgtggtcttttggatctatttggtcagagagtcagtctttggtttcttgc 39481 cctggaaactgcttgttgctacttgtggtgggggcagcatttggaagtccaggtgctctg 39541 cccacaaactttcaacccatcatttgtttttcatccctttctcattgccactttgtgtgg 39601 tgcctgggacttctgggacctatagttcaagggtcatatataccaatggctcacatgaca 39661 gcactgatcactctgccagctctcctctctttgcaaaacttatttcagatttttcatttg 39721 acaatacctttcctccagttgtctttattcttggcagcatatgccttgtaacctttaaaa 39781 aggaaggtaaataatttgagaaaaaatgtaccaagtcctcagtgatacattcttactaaa 39841 gactcccagttttaacaaggagttgggctggagccatggctcaacagttaagagcactac 39901 ctgctcttccaaaggacacaaattccattcccagaacccacatggccccttccaaacatt 39961 gataactctcgttccagggcacctcatgccctttcctggcatctgagagaaccagcataa 40021 acatacatgcaggtgaacattcatacacataaaatgaacattaaaaaagaaatgaaatag 40081 agaaagggtttacataactatttaataactaagactgcctaataatgtagggacccataa 40141 agaaaatctagtaagtttttacaagattccactcaatcagaccaaacattactgttactg 40201 acagagtaaaaagtcacttccaatagtccaagaacaactttgtttcatttctcaggcact 40261 gtctgttttgtggcatatgtgcatggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg 40321 tgtgtacaggtgaatgctgctcgtgtatgagcacatgcaggtgtgtgtttgcatggtgtg 40381 tagacagagtttctgacctgcctggtcccacagctgtttggccacaaataaacatacaga 40441 ggcttatattaattagaaactgtttggcctatggcttaggcttctcactggctatctctg 40501 tcttaattattaacccataactactaatctatgtatttctacgtggcgttatcttaccgg 40561 agaatacttggtgtcctatcttctcagcaactacatggcgtcttctctctgcgtcttctc 40621 cccagaattctcctcgtctggttgccccgcctatactttctacctggctactggccaatc 40681 agtgttttattcatcagccaataagagaaacatatgtgaagaaggacatttccctatcaa 40741 tggtgtgtgtgtgtgtgtttgtgtgtgtgtgtgtgtgtgtgtgtgtatgtgtgtacatgg 40801 gtatgtgagcacatgtgggtatatgggtgcatgtgcacctgtgtgtgtgcatggtggcta 40861 gagttgaggttagatgtcttccttggctgctctccaccttttttttattgaagctctcac 40921 tgaacttagagctcactgattcagctagtctagctacccggcctgctctgggggtcccct 40981 gccttcactttccatgtggctaccatatctactttacatttatgtgggtaatggggatct 41041 gaactatggggtcctcatgcttgcatggcaagtgctttatggactaagacatctttctag 41101 cctttaccttttttttttttgaaagagtttttttttgctaactgggaactcaacaccaga 41161 tagctagtctactggtcactgaggcccagggatctactatttctgcttctcttcccaagt 41221 gctgggactacagactgtaccaccatatccatatttcttttagcatgagctctggaagtc 41281 aaactcaggtcctcacgctcacaaagtaagtgttttatctaccaagccatcttcccatct 41341 ctgttgttttaaaaggctttgaatatgggatgtgatgaagggaggtgaaattctgagata 41401 aatttcttgaaaagaagaatgaatcaagtaggagaacctcctcctggtgctgtctttcag 41461 ttccatgtccacacagcataaacattatgattatcattccacagattgtaattagtcttt 41521 ctctgttttgccagtctgctcccaaaaaatgacacagagagacttcttattaatgatgaa 41581 agctttgccttagcttaggcttgtttctaactaactcttgtaacttaaattaacccattt 41641 ctattcatctacctgctgccacgtgattcatgacttttacctctctctcattctgcatat 41701 cctgcttcctctgcttctggctcatgatcccgcttttcttcctctccgagtgctctgtcc 41761 ccagaagtcccgcctaacctcttcctgcctagcaattgcccatttggctctttactaaac 41821 caatcacagtgacacatcttcacgcagtgtaaaggagtattctgcaacaacaggtgatga 41881 agccaacattccaagaggccagggcttgcctagggcacatagctaacttaagaaaattag 41941 gatcgcattctacatctgtctgactctgaattggatctgaactgtgacttgcatggaaga 42001 cccaaagaccctgagaaagtacaatgacaaaggggctgactctgtccacatggtgttagc 42061 ccaggtttcccacaggaggaaaacccatcctaggcaagagaagtggtcttcatcaaacac 42121 tctatgaaaagcaaatcagactcaaatgtcaggatttgtgctttacagatcgatccggta 42181 agatgaaagaacttcctgaaagtgtgtgaaggcctaaagtcagggctgttcatggaaggc 42241 actgactacagaatgaggtgccagaagcctagtcagagcctctagggaataaagtgtcag 42301 atgatcttctaaaaaagttgaagtttcaccagtaacagaatggccccactattaaaatgt 42361 gagcaaactcagaagtcattgtagcatatagaagcacagacctatggattgctggatgga 42421 gcccaggtattcactccatcctgaatagccagctggggagctagctcagtcagttaagta 42481 tttgctatgcaaatctgaggaccagactttggtctcctgcatccacagaaatggtgcaca 42541 cttgtaatctcagcactggggaagcagtcagccagatccaacagctgcctagccagcgga 42601 aacagccttatcagaaactcatgggtcctggtgaaagatattatctcaaataacaaggtg 42661 ggaagctcctgaaggacactggaggttaacttctggataaacataggctcgccccaccac 42721 cagtgagcatgtgcctaaatccgtacataacaatgatgtaaagatggaattcattccagt 42781 gaaaagtaagcctcctggactctttttttttttttgttgctagatattctcgagacctca 42841 ggagagaaggtttgccatcatctatataacatggtactcaacttccctgtagtccacaac 42901 attcctatttctatatgatggagaagaggccactgcccctcccagacatctcagtctcaa 42961 atttgttaccagttccctctcctaataagtgcttagggttagtgttgtagagaagggctt 43021 tacatgaagtgtgtgtgtgtgtgtgtggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg 43081 tgtgtgtgtgtgtgtgtaacctaaaggctttccatgtttccacactgaaaggttcttaag 43141 actgagaacaaccagataagagtccaaattctagaaaccatgggaaagtgtaatattgaa 43201 agtcagaacaaggcatggtggtgctcaccttgaaacccaccacttggggcagaggcagtc 43261 agatctctgtgagttcaaggcccagcctggtctacagactgtacatagtgagttccaggg 43321 ccagaactacatagtgagatcttgtctggccaaaaatatataagtaaataaaataaatca 43381 gtacatggtaacttgttcttatttcagtgtctgtttctcaagcatgactttggcttaagg 43441 atttttcccaacttgtttttgtgattgccactgtatcatttctttgtgtgaagttactaa 43501 gtggtttctgtatttgatattatgttctgacctagtttcttttcatattaaacccatttg 43561 tatatgaaaactgcaaagaagtgggttttttgttttttgggtttttttttgtttgtttgt 43621 ttgtttgttttttcttggtgttctcatgtgacctttccaatgtttgcttccagaatagac 43681 ctgcaagttgggatccacactgccatctgaagtcctgcaccccaagtttcaggtatgttt 43741 tgatggcagaatagcttttctagactgtgacaataggggcataaagccacaaagcattcg 43801 ctttcctacaggttatgcacccactctctgagtgattggctgtgcatcatgaatattatc 43861 aaaatggaggcagttcagtttggagtgctgtcttttatgcgcttattcatggcaatgcca 43921 atggaacattcggcaacatatactactaatcatgcatggtaactgaactgtgttgtgcaa 43981 ggaagacctcatatgacctacctttgcatatgctgaccttttctgtgacagactcctata 44041 atactgagagtggtactgtatggaagagtgtgtgaaaatgtattgtttaaataacagaca 44101 gatgcctctaaatacaacacccaagcagagaaatggagcatcactggcactttggaggcc 44161 tctgggtaacctttccagatcacactgttttccttcctccaccaataaccactttccctt 44221 tggatgctactcatagttaacatctttacttttgttgttgtcccactgatgctaagaaaa 44281 ataacttcaactagcaagcacaacactagatgaattaagagtgatattgactgtgtgtgg 44341 tgagtctcagaagactagctgcctcaggattcatgaatgcttacaggaaccctttagcaa 44401 ggtcaggaatgagtcttaggatccatgtggctcatagtctccagcctggacatggagtag 44461 cacagtgtctgagtgccccaagggaatgggcttgttcaggctcccctccccgtccccagt 44521 tccaacaggtctcagatccaggacatcagagctgagtgaagagcagagctaaaaggagca 44581 ccatcggagccctagaagcagaatagggggggacacagcacacagagacaagaactgagg 44641 ccaggctgctgtgtgctttgggcctaagttgacagatgaaacatggtagggtgaccacat 44701 ggaggatgtctgtgcacatccatcaaactggcaggtccccccagcattttctgggagctt 44761 ggggtcctcttttccatgatcttcagcttctgtattctatgtgcgctgttaccatttcat 44821 cttggtagagtctatccttctgttatttcttgagagtatgtcccaattcttgcctggagg 44881 tttggctaaatatagaattctaagcagagggtcatttctccttcagatatttaaagacac 44941 tttctgtattgtgcctcattgccattgttgatatacctgaatctaaattgatcccttggt 45001 gcgtgacttatccccacagccaagggccccttcccttctggtctgtgctctggaagtctg 45061 caggcacatggtatgggtagccactgtttcattcatagttcaatgctccgataggccctt 45121 ttgatttgataactctatccctttcccccattcccgttgatgatttcttcttttgttccc 45181 cttttgatatagtttccttgctgatgctgtgctaaaatattcctaccaaaaacaacctgg 45241 ggaggagaggcttcatttggcttacaattccagctcacagtcattgagggaagtcagggc 45301 aggaactcaaggcagggagcatggaggaattgcctgctggcttcctctctgacttactca 45361 caggttcttgtaggctagctttctgataacatctcaggaccacctgcttagcaatagtgt 45421 ggtccacagcaggtttgaaccttctgcatcagttactaatcaagacatttgcccaaagac 45481 atgcccacaggccagattgatgtaggcagttcttaaatcaagtcttttttgtcaagtgac 45541 tctagactgtcaagtcgacagttgatgctaactaggacactattctaccacttttcttgg 45601 tagaaatattattcggatattggagttcttggactagtttttctggttctccttttcttt 45661 cttttcctgttatttatatttgttttatgagatagggtctctctgtgaagttgtcctaga 45721 ccttctggccctcctgcttataattcctaagaactgatattacaggcaggtgccatgagc 45781 ccaacgttttttcttttcttttcactgcactctgtttgagagtctcatcgtcacagtcat 45841 tcacatcttctattgtcttgtttttctttttaaatgtgcattggtgttttgcctgtatgt 45901 atgtctgtgtgagggtgtcagatcttggaattacagttccaaataatatttctaccaaga 45961 aaaagtggtagttgtatcctagttggcatcaaatgtcaccttgacagccttgagtcacct 46021 gagaagaaagacttgatttaggagctaccatgtggttgctggtaattgaacccaggacct 46081 ctggaagagcacccagtgctcttaactgctgagccatctctctggcttccttctattgac 46141 ttttgcaggcttctttcttgttcttttgcaatttcatggtctctgactgttcttcacaga 46201 ctcttacctcatgcttaagatgtctcttactccttcaaggatactgagtttttgaagttt 46261 taattctcctgactactgtcttttccctcctgtttgtcattctctgtttgccctggcctc 46321 tgtctttcatgcaggaagacttttcatttgcttttaggtttttattttaactattggttc 46381 atgactaaagggctagatgaaaaggccagtgagaaggctggagcatatgggtgatacttg 46441 tcaaccgggagcctcactgtggaatgcttcagtggcatgtgaaatcctgtggtatttgct 46501 caggcaagtgcagctgttgaatgcagaccagagcagcttccttcgaaggagtcagatgtt 46561 gctgactgtctttctgcagctggtcaggaaggtgggatagacttcagctcttttcaaaca 46621 gtggtcaccaaacaaccacttgcccagagactttgtgctttaccattctcagagaacaga 46681 cctctggatggccccatggtggaagcagcgcacctgtctatcacaggtgctctgaaggag 46741 ttggaagaactacccattgtccacatttcccacattttcacatgccagcttcactctggg 46801 atctgggtgacagtggggctgacataatggcaggggttgcagtttcagactcagagtatg 46861 tggtaggaatgctgctgtctgagggaagactcatctgagcagtggaggctttgcctgttc 46921 cctggcatcatttgacctgcccctccttagaactgggaaccccagttctaaagctccctg 46981 ctttaaagattctgtgttggggtaagttcttagctttctcaggctaggtcctctgctctt 47041 gggtttccacggcactgttgttttccctctggctttgtgagtggttgtcttttgaaaaac 47101 tagttagtttggaaaattttgggagggagtcaaataagatgtatgcattttgccatgtaa 47161 gtcctaaccaagccatctgctgtggtattttcctgagtttggttctgcccctataggcag 47221 agtctgtcatcacagataattgcattttgaacttgagcatctcccttccttctttgtctg 47281 cctgaaaaagtctctttataaaaaaatgtaatgttaatttaaaaagtattcattattctt 47341 gtgttgtgatacatgagtatatatatgctatgatgcatatgtgcaggttggaggacaact 47401 ttctgtagttggttctctctttctcccttcatgtaggttctggggatcgaacccaagtca 47461 tcaagcttgcacaacagcacctttaccttctaagccttctcatcagccctttttttattg 47521 attgattggttgattgattgattgattgatgctagggatagagcctagggtcttttacat 47581 gctaagaaaatgctctaccactgaactgcactcctagcccaacctgctaaattcttacac 47641 tgtcttcaaaaagaagctctgatgctggattctgcaaagtccatttttatccctaaattc 47701 ctaaagctgtttaaatctcgtgagtcttactgtacagaccagctctgtgcaccatcttcc 47761 acaatctccatgacctcctcaggatgggctggtatctctgcagctctgcccagtgcctac 47821 caggaacttacaggtgtcaccaatgaatttattggtgcatgctcacttcatcttgtccct 47881 atccactttctgctttgactccttctggtaagagacaagtgtgttaactacttgtgctat 47941 caccacacagaaatccatatcccataatcttagtcctttttatttacttatttttgagac 48001 agggtcacactctgtagctcccacactggccttaaacactgacctcgaactcatggtgat 48061 tctcctgcctaaacttctcaaataccatgattacaagagtgacacaccatgctgggagtc 48121 ataatcttaagtttaaaagtgagggactggtcagtttactgtgctaggttgacattgtat 48181 agaaatgaacagccatgttggtctggaaatgttcctagttttcatttgtacaaggatatg 48241 cagtgtgtgaaatagggagagtcttacctatgtgggtttgatcacagcaattaataaaat 48301 atgctctaaataatgaaaaaagccagtaactagtagtgtttctgaatcctcactaaagct 48361 ttaatacatcataaataatatatcactgcagattatgtctacatgttatacatatcacat 48421 ttatagtacaatctgatctttgtcacctactgtaagcacaactgaaaaacaaattttctc 48481 atagctcaatattaagtcattattatccccataataagtaattattatccccataatgaa 48541 actatctattgagggagtcagaatctgagatagttaaataaatttaagcatgtattttta 48601 gtgtcaatggtaaaaattaaatgttcataaagcctgtatgactccttttaaagtagtttt 48661 aattttatgtgtatacatatatgcatgttttgccttcttgtatgtctgagtaccacttgt 48721 atgtctggtgcctgaggaggccagaacgtatcagatcccctgaaactggtattacagttt 48781 tgagctactatgtggctgttgggaattgaacctggatgctctgaaagagcagccagtgct 48841 cttaatgactaggccatctctccattttcttaaaaaaaaatttaaaacatttactctaag 48901 atttacttttatgtaggtgcgtgtgtgaatgtgtatggtttatgcattggggtggggagg 48961 atggattagcacagtcacagaagactagaggagggtctctactattgctttctgtcttct 49021 acccttgagacagggtctctcactaaacctgaaactcacctttgcagctggggtagctgg 49081 tcagaaagatcctggaatctgtctttctccctggccctaatgcttgagttacaggcccat 49141 gtgaccatacctgtcgttttactggggttctacagagtcaaacccaagtcctcacgcttg 49201 catagccagcgattttaccgactgagacatttatctgccccaattcataattcttctctg 49261 cttccattaataatcccatctatgtccccttcatacatatttctgaaatagacaaaatga 49321 atacaagttagacatcgagtctgattaatcttcaacttctttgataaccaggtattgatt 49381 tctgacttttgaagatggatgaaggcacagaagtctccactgatggaaattccctgatca 49441 aagctgtccatcagagccggcttcgcctcacaagacttttgctcgaaggtggtgcttaca 49501 tcaacgagagcaatgaccgtggcgaaacacctttaatgattgcttgtaagaccaaacaca 49561 ttgaccagcagagcgttggtagagccaagatggttaaataccttctagagaacagtgctg 49621 accccaacatccaggacaaatctgggaaaagcgctctgatgcacgcatgcttggaaagag 49681 cgggcccggaagtggtttccttgctgctcaagagtggggctgacctcagcttgcaggacc 49741 attctggctactcagctctggtgtatgctataaatgcagaagacagagataccctcaaag 49801 tcctccttagtgcttgccaggcgaaaggaaaagaggtcattatcataaccacagcaaagt 49861 caccctctgggaggcataccacccagcagtacctcaacatgcctcccgcagacatggatg 49921 agagccatccgccagccacgccttcagaaattgacatcaagacagcctccttgccactct 49981 catgttcttcagagacggacc SEQIDNO:3(Chromosomalregion5,000-55,000basepairsdownstreamofCHOGS genecodingsequence) 1 GGGCTCAGGCATTTATCGTTCAGAGATTGACTGAGCTGTAAAGATGGAAAGACAAACTTT 61 TTTTTTTTTTGATTGAGTCGGGGTTTCTCTATGTAACAGCCCTGGCTGTCCAGGAACTCA 121 CTCTGTAGACCAGGCTGGCCTTGAACTCACAGAGATCTGCCTGCCCCTGCCTGTCGAATG 181 TTGGGATTAAAGGTGTGAGCCACCACCGCCCCGCTGACAAACTAGACTTTTAGAATGTAT 241 TATGAGATAAGGTTTTGTTATGTTGCCCAGGCTGGACTCAGATCTGTAGCAATCTATCTG 301 CTCCAGACTCCTGAGTGCTGGGATATACAGACCTGAGTTACCTGTACAGCTTTCTAATCA 361 TCCCCCGCTCCCCCAGAGACAGGGTTTCTCTTTATTGTTTTGGAGCCTGTCCTGGCACTG 421 GCACTCACTCTGTAGACCAGGTTGGCCTCGAACTCACAGAGATCCACCTGTCTCTGCCTC 481 CTGAGTGCCGAGATTAAAGGTGTGCACCACCAACACCCTACTTTCTAATTCTTAAAGCAA 541 GGCTCCCAACTCCTCCCTTGTGTGTAATCAACAAGGTTCTTAGACCCTGTCTGCAGTGTG 601 GATTCCCACTAATAAGACAGTGGCGGCACAGTGCTGTGTGGCAGAGCAAGCGTCCATCTA 661 GTTCCTATTGTCATTCTATGATTTGCTCTTCTGGGAGCCTTGTCATTCAGCAAGTTCCTG 721 GGCTTGTCTTGGGATTGCAATGTGCCTCAGCTTGGCTAGTTCCTCTGCGGCAGAAGCAGT 781 GTTTGAACTCAGTGGGCACTCAGTCACTACATCTAACTTGTTTGAGGGCTCTCTGCATTT 841 GCTTTCCAATTAAGGTTTAGGATGACTCCTCCCTGTGACTCTTATCATCCTGCCTATTAA 901 TGCTAAATTAGAGAGGCATTCAAGATAACTGCCGAAGATCTAATAAATAAATGGGGTGGG 961 TGGGTAGGACTATAAACCAGTTTATAGCATGCAAGAAAGCTCTGAGCACCACATTCAAAA 1021 ATAAAGTGCTGTGAGCCTGGTGGTGGTGGCTCACACCCTGATCCCAGAACTCAAGAAGTA 1081 GACAGAAGGCTCAGATTCAAGATTCAAGTTCTTCCACTATACAGCCAATTTGAAGTCAGC 1141 CCAGACTACATGAGACCCTGTCTCAACTAAGCAAATGAAAGCAAACTGGGGTCCAAATAG 1201 GCACTATTCGATGTTTTGATGCAAGTTTGTGACTGAGGAGTGGAGGTGGCAAATGAAGAC 1261 TTTTTTCTTCCTCTTCTTCTTCCTCCTGGGTCCCGTTTTTTTTAGGGTGTTCTTAGGATA 1321 TGTATGTCTCATTGGCACTACTAAGAAGTGTGGGGTCTAGGGAACTTCCTGTTATGTATA 1381 CAAGCTAATCTTCAAACAATTGTGTGGGCTGTTTTGGTAACTACTCAAATAATGCTATAG 1441 AAAATTGTACAATATATTGGGGAAGGAAGGGAGTTTTACACAGGAGTCAACATGACTCTT 1501 GTCTCTGGAAAGCAACTTGTGATCCAATGAGGAGCTAAATTTAGAGACACAATTCAGGAA 1561 GAGAATCCAATCAGAGCTTCCTTGTAAAACAACTCACCTTCACAAACAAGTTCATTCCTA 1621 ATCGAATTTAAGGTCTAGAAACTGCCAACCTATTAATGTTTCTATAAATACACTTGGGGT 1681 CAACTACGTAGCCAAGGAAATCTTTAATAAATTGAACACAAATTGTCAGGGGAAGGTTAT 1741 TGCTGGGACTCCTGGAAGCATGTATAAGCAGGGTAGGGGTGACATAGGGGTGGGGGGCAG 1801 TTAACTCACAGATATTAGTCTCAGATATTAATGGCTTGTGTGTGAGCTGTCTGCCACACT 1861 TAATGTCAGTCACCTTGCCCGGAACTATTTTTCTCTCTGATTCCAAATGTAGCTATTGGT 1921 CTATTAAATGATTAACTTCCACAGAAACTGATAATATCCTTATGGAATCTGACTGTGGTA 1981 AGCCTGTACACCCCCGCCCCAATTTCCTTCTAGATTTAGAATTCCATTCCATGAGCCATC 2041 ACACCCACGCTGAAAAAAGAAAACCTGTTGAATCAAATTTGTGTTTTGGAGGGTAAGAGC 2101 CACCCTTCCAATTTATAAGGCTGTCTATTTCTTTGGGGGGGGGGAAATGAACCAGTATCT 2161 TCTATTAGTAAAAGGAGTGTTTGAGCATGGGCACTACAACCCACTTCTTTCAGGGAGATT 2221 CATTTTTCTCTGAGAACTCAGCCTCTCTGTGCTGGTGCCACAGGAATTCTTAAACTCTTT 2281 CAACTCTCCAATTAACCAGAGAGCAAACCCAGCACTTTCCATCTATGAGAAATCTACACC 2341 ACTCATGGAATCATTGTGTGCCCTCTCTCACTGCCTAACAGGGGTACCCTTGCCAAAGAA 2401 AAGCAACTTAATGCCAAAAAGGTGCATCACCTGGCACTGCTTCCGAGGATGGGCAATGTG 2461 CAAGCACTTTGTTCAGTGGCTCTGCCTTGGGGTCTCTTGAGGGGCGGCAGGTTACCTGGG 2521 GTGGGGGCGCACACTCTCTGAAGGTGGGCTGCGTTCAGTTTCCTGCTTCAGGGGCTCCTT 2581 CATAGTACCGCCCCCTGATGAGTTTCTGCTCAGACTGGAAGGTGTCAGGTCCCAAAGAAA 2641 CCTGGGACAAGGCTCACTCAGTACCTGTCGCTTCTCCCAGCACGTCTCACCCCACCCCTA 2701 CCCTAAACTTCTCTAGCCCAGAGGCTGGGCTCCCCCTTTCTCTTTCCTACATAACCCTGC 2761 CATTTTAGCTGTGAGCTCTCTCCGTCTTTAGCTCCTCTACTGTTCTTTTATCCTCTCTTT 2821 TCTCTCTCCTCTTCTTCTCTCACCCCCACCCCCACCCCCATCTCTCCCCCCATGGTCTGG 2881 TTCAGTCTGGACCCTTTCAGATGCCTCTGTCTGAACTCTCCCTCATATCTCAATAAAACC 2941 CTTCTCTTCAGCCACGCCTTGGAGAGGTCATAGGCTCATTTTCGTTCAGAAGGCCTATCA 3001 AAGAATCTGTGGGCTTATCTTTACATTCACAATAGGCAGCTTGGCCCTGAGACCACAGTC 3061 CAGGTTAAAGTGTTACCTTGGAAAGAAAGTCTTTTATTCAAGGTGTCTGGTTTCTTTTCT 3121 TGTTTTTGTTTTTGTTTTTGGAGACAGGGTTTCTCTGTATTATTTTGGAGGCTGTCCTGG 3181 AACTCGCTCTGTAGACCAGGCTGGCCTTGAACTCACAGAGATCCGCCTGCCTCTACCTCC 3241 TGAGTGCTGGGATTAAAGGCGTGAGCCACCAACGCCCGGCTCAAGTGTCTGGTTTCTTTT 3301 GATGTCTTTAGTTTCTTTAATCCCATAATTCCTTTAATTATACCCTCTTGTCTGTCGGAG 3361 AATGACATCAAGGATATCCAGTTCAAGGTTTCCTATGTAGTTCAGTCATAGAGTGCTTGC 3421 CCAGCTGCCAGACTCTGTCAGATGCCCAGCACCACACACATACAAAGCATTTCCAGCTCT 3481 GTGTCTGTGTCAATTACTCCTGTCTGCTTCTCCATCCCCAGACACCAGGAGGGCCCACAA 3541 GAAGCTTGGAGCAGGGAAGAATAAAGAGACAATATCCATAGACACACAAAACCTCCAAAG 3601 TACTTATGCATTGAGGAATTACAGCTTACAAATCCAGTCACAGTATCTATATTCATGTTA 3661 GCCTGATTTCAATCCCCCAGCTACATATTCTTCCATGAGCTAGCTCCTTTCCTATTCAAG 3721 ACTCCCTTGATAATAGTTGTTATCAGACTTTACCCCTATTAAAATATTTGGACCGTTTGA 3781 GAGCAATAGCTCACCTCTATAATCTAGAACCCAGGAAGTTAAAACAAGATGTTTGCTGCA 3841 AGTTTGATGCCAGCCTGGGCTACATAGCAATTTCCAGAACATCCTGAGCTACAGGGCAAA 3901 ATTCTATCTTAAAAAACAAAAAGTAGACAGATCAGGTGTTTCACCTTGTTTCAAAAAATG 3961 CAAAAAATATTTTTTAATTGTAGAAATATATACGCTAATTCCTTTGGTACCCTAGGCCAA 4021 GTGACTAGATGGGTTAGTCTTCCTTCTGGTCCTCACAGAAGAAAGTTAAGTTCTCAGCAG 4081 GAATAATAAAAAATATTAAAAAAAAAAACAAGCTGCAAAATTCTGTTGTGGTTCTGCCAA 4141 AGTGTTCTCAGGAGTGAGGGCATACTGGGATTTAGTCAAGCAGATATTTCTGTTTGAATA 4201 ACTAGGATCTGGGAGCCATGGGACACCACCCCCACCCATAAGGGCTACTGAAAACCACCC 4261 CTGGAAATCTGTAAATATTGCTAAGGCTCTACCCTTTTGCTCAGAGAACAACCACCCACA 4321 AGGATAGGGGATAAGTTAGTTCTGTAGTAGAGTGCTTGCTTAGCACACAGAAAGTCTTTC 4381 TCTCTCTGTCTTTCTCTCTGTCTCTGTCTCTGTCTCTCTCTCTCTCTCTCTCTCACACAC 4441 ACACACACACACAAACAAACACATGAGTGCACAAGAAACTTCTAGGTGCTACTAAACTAA 4501 TGTAAAATCATGCAAAGTTCATAGAGAATTCAACAGCTAGTGACAGGATGACCCGAACAC 4561 AAGATTCTGCCCTAGTCCTTGTATTCTGTAGTCCCCAGTTTCTCTTTACTGCCACAGTCT 4621 CCTATCTCTGACAGCCTCCCTCTTTGCAGATCTGGCAGTTTCTGGGCCTGGAACTGCTTT 4681 GGTAGAATGTCTGTACAGCATGCACTAGGCACTGGGTTTGATCCCCAGCACTGCATAAAT 4741 CAACTTTGATGTCACACCTATAATTTCAGCACTTGGCAGGGATCGAAGCAGGAGGATCAG 4801 AGGTGAATCAAGGCCAGCCTGGGCTACTTGAAACCCTGGGGAGAGGGATAGAAGAAGGGG 4861 GAGGGGGGAGGGAGAAGAAAGGAAGGAGGGGGAGGGAAGAGGAGAGGAAGAGAGGAGGGA 4921 GAGGGAGGGAAACAGGGAGGGAGGAAGAGAAGGAGGGAGAGAGGGAGGAGGGAGGGAGAG 4981 ACTAGTGTAAGCAGAACCTGTAAGTTCTCTCCTCAGCCTCAACACACCCCAGCTCCCTGC 5041 TGTCTCCCGGTCCAGGGCTTCAGGGCCTGGCAGGACAGGCAGCAGGTTGTTTTGCTCTCA 5101 TAAAGCCATGTTACATAACTAACTAATGTTTTGAGCAGTGGAGCTGAGCCAATCTAGGTC 5161 ACATCAAGAGGGAATGGGGAAAGAGGATGATCACGGAAGTGGTGAGAGGAAGGGAAACAA 5221 GAAGGGAGGAATAAAAAAAAGAGGCGAGAGTGGAAATGGGGTGCGATTATTTAATATCTG 5281 CTGCCTGTTCATAGTTCCTGGTCCTTAGGGACAGCATATATTATCCTGAAAAGTCCTCTC 5341 TCTATTTTATCTAGGCATTCTGTCATCCTATAGCCCCCACTCTGGATGGCTGAACTCTGT 5401 GCCAGCAGCCTGCAGGTATCACCCCTTATTGGAGTGAGGTCTATTCCTTATTGGAAGCAG 5461 TGGCAGGCTGGTAGGAAACAAACAGGCCTGGTGTTGTGGAATGCTGTCCTCCCAGCATGA 5521 CCATCATTAGACCTTATGGAAGCAGAGCGAGGGGGGCATTGTCCTCCTCCCCAGGCTCCT 5581 GCAAGCCTACTCAGCTCAACTGGTTCCCCGGGCCAGACTTAGGTGCAAGAGTTGCTTTGG 5641 TTTGTTATTGGTGGCCTGTGTAGCTGAGTAGACACATGCTCACCTACATGATATATGATG 5701 GCTTGCAACCTTCTAAAAGTTCAGTTTCAGGAGATCCAGAACCCTCTTTTGCCCTCCAAG 5761 GACACCAGACACCCATGTGGTACCCATACGTACATGCGGGCAAAACACTTGTGCATATAA 5821 AATAAAAAGAGATGGCTCCGTGGCTAAGAATGCTCCCTACCTCCAGCTCACCCACATCTT 5881 CACAACTGACTGTGAATCCATCCATGGTTCTCTTCTGACCTCGGAGGGCACCTGTGCCCA 5941 TGGGGCATACACATACACATACACAAAACAAGTATGTAAATAAATAAATATTTAAAATTG 6001 GGGCTGGAGATGGCTTAGTGGTTGAGAGCACTGGCTGATCCTCCAGAGGTCCAGAGTTCA 6061 ATTCCCAGCACCTACATGGTGGCTCCCAATCACCTAAAGTGGGACCTGATGTCCTCTTCT 6121 GACATAAGGTCATACATGCAGATAGAGGACTCAAATGCATAAAATAAATAAATAAATCTT 6181 TAGAAAATAAGTACATAATAAATAAATATTTAAAATGACCCAAATTAAGAAAAAAATGAA 6241 GCCAGGCAGTGGTGGTACACTCAGAAGGCAGAGGCAGGCAGATCTCTGAGTTTGAGACCA 6301 GCAGTTCCAGGACAGCCAGAGTTACACAGAGAAACTCTGTCTCAAAAAAAAAAAAGAAAA 6361 AAAAACAGAGAAAGAAGAGAGGAGAAAAACAAGAACAAAAAATAACAAAACAAAAACATG 6421 GCTTTCCCTTCATGGCATCTGCTTCATCTGCCTATTTGGTAATGATCAGGGCACTACACA 6481 CCCAGTGCTTCATACCCTGGCCATGTTTCTGTTCTTGGTGTCACCACCAAGTTTACTAAA 6541 GATGGTTCCAGAGTGACATTAGCAGCCCCACACCCCAATTGCAGCTAGCAGTTGAGGAGA 6601 TTTCTGGCTTTTTGTCTAAGAGGAAGGTTCTTTGGCTAGGAGATATACTGAGAAGGACTA 6661 GGAAAAGGGGTGTCTAAGAAACTTGGAGAGCACATTTTTCAAGTCAGAAAGAACATAGAC 6721 ATATTCTGGGGGTGGGGGTAGTAAGATAATGGACCCTCCTAAGGGAAGGATTGTGGGGTT 6781 TGCCTGAAGGGGCTGAAGCAGACCACTGAGCAGGCCAGACCACCAGCAGCTTTTGAGAGG 6841 TGGGAACACTGCAGCTGAAGTCACTTGTCACCTTCCCAGGTAGTTCTTACTTCCAGCTCT 6901 GGCAGGGCTAGATAGCCTAGGAACTCCCAGATAGGAGTTCTAGTTCTTCTTCTCCCAAGC 6961 TGACAGAACGTGAGCTCAGAGTCTAGGGACACTCCAGGTTAAGGACGGGGCCATTCTTGA 7021 TTGTCAGCACAGATAGATTTTAATTAGAGAGCAATGACATGACAGATAAACAGCCCCTTA 7081 TCTAAAGGGGTACATCCCAAGACCCTGGAGGACTCTTGAAAACCCAGATAGGAGCCAGCC 7141 ACGGAAGCATATACCTTTAATCCTAAGATTTGGGAGGCTGAGGTAGGAGGATCTCTGTGA 7201 GTTTGAGGCCAGTCTTGTCTACAAAGTGAATTTTGGGACAGCTACACAGAGAAACCCTGT 7261 AAGAAAAAAAAAAAAAAGAAAGAAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGG 7321 AAAGGAAGAAAAAGATAAAGGAAGAAAATCCAAATAGGAAAGAATCCCATATATACCATA 7381 TTTTTCTTAAACATACATAGGTTTATTCATTCTCTCTGTGTCTGTGTGTCTGTGTGTCTG 7441 TGTGTCTGTGTGTCTGTGTCTGTCTGTCTGTCTGTCTGTCTGTCTCTCTCTCTCTCTCTC 7501 TCTTTCTCTCCCTCTCTCTCTCTTTCTTGTCTCATAAATCTCAACACTCAGGGACCCAGA 7561 AGATATCCCAGTGGTTAAGAATACACACTGCTCTTGCAGACCTAAACTCAGTTCCTTGTC 7621 CCTACTTGGGGCAGCTCACAACCACACCTGTAAGTCTAGCTCCAGGGAATCCACACCTTC 7681 TGGCCTGTGCAGGCACCTGTGTGAAGGAGCACATATCCTTCCCCATAATTAAAAAACAAT 7741 CATTGAAAAATAAAACTCAACCCCCTCCCCCGGGACTCAAACCAGAGGTAGTCTCCCTGC 7801 CGTAGGCGCTCAAAAACTGGACTTTCAGGTGTGAGCCTCTAGGCCAGGCTGCTTTTCTTA 7861 ACTGGCTACCGTGCTCTTGCCTGAAACTTCCAGCTTGAGACCTCATAGTAAAAAGAACAT 7921 ACACGTCTTCTGTCTGTACTATTTTACAGACGGCTGACATGTTCATACCACGTATTTTAG 7981 CAATTTCAGCACTTGGTATATTTTCTGTCATTCTCAAATAACTTTCACCTTGCCACTTAG 8041 GGCAGTCCAAGGCTCCTCTTAGATATATCCAAATTATCAGCCACCACTTCTGCCTTTACT 8101 AAGTAAGACAGGGTACTTAACATGGAGTACTTAACACAAGCACTGTGATCTGAAGGTGGA 8161 GACTGCTTGCTACTCAGTCACAGCTTAGCATTGCTAGAACAAATCCTGAACAAAGGGTAA 8221 TTCATGACCCAGGCAGGGCAGAGGCGGATGGCTGTTCTTGCTCCTCAGAAACCCCTGTGT 8281 ATAATTTCAAGCTTAGGAGTTGTTTGTCTTTGGATGGAGAGGGTCAGACCTAGGGCTTCA 8341 CTCACACTAGGCAAGCACCGCAGGTCTACCTTCGAAGAGAAGAATTTTCACTTAGCGTTT 8401 TCAGATATAGGTCAACCTCAGCTGGCTGAAACTTTGACTAAGTGAGCAACTGTGAGGGTG 8461 GGGAACACATGCATGCATTTCTTCATGTTATAACATCTATTTATACATAAACATATCATA 8521 TAAATATATTCTATTGCATATAAATATACATAAATGCACACTCATGTATAGATATCAATC 8581 ACATAATTTATGCTTTTATTCATAGATTATCTCTGGGAGGTGTACAATTACTGACAATAC 8641 CTGCACATGATAGTACACGTTGTTCTAGTTAGGTTTCTTTTGCTGTGACAAACACCACAA 8701 CCAAAAGCAACTTGCAGAGGGAAGGGTTTATTTCAGCTTACAGTTGTATTCATTATGAAG 8761 AGTTGGGAAGTCAGGACAGGAACCTGGAGGCAGGAACTGAAGCAGAAACCATGGAATAAT 8821 GCTGCTTACTGGTTTACCCACCATGACTCAACCTGCTTTCTTATATCACCAGGACTGCTT 8881 GCCCAGGGATAGAACCACACATGGGGACTGTACCTCCCACAACAATCATTGATCAAGAAA 8941 TGCCCTAGAGTCAGGGATGGTGGCAAATGCTTTTAATCCCAGCACTCGGGAGGCAGAACC 9001 AGGCCTTGACTGTGAGGTCAAGGCCAGGCTGGTCTACAGATTGAGTTCCAGGACAGCCAG 9061 GGCTACTCAGAGAAACCATGTCTCATGGAAAAGAAAAGGAGGAGGAGGAGAAAGGAGAAG 9121 GAAAAAGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAAAGAAGAAGAAGA 9181 AGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGTAGAAGAAGAAGTGTCCACTGGACA 9241 ATCTGATGGTGGCGTTTCCCAATTGAAGTTCCCCTTCCAAGATAACTCCAGGATGTGTCA 9301 AGCAGACAAAAACAAGAACCAAGACACATGTTTATAATCCCAACACTGGGGAAGTGGAAT 9361 AAGAGGTTTGGCAGTTTAAGGCCATTTTCAGCTACATAGGGAGTTCCAGACTATCCTGGC 9421 TACATGAGACCCTGTCTCAAAACACCAAAATGCAAGGGAAAAACAAAAAGCAAAATAATG 9481 AGTACAAATAGCAGTGACATTCTGGGGAGACAGCCTGGAGGGGGGGATTGCTTATTATCT 9541 CTCCCTACCGTTTGGAGTTTTTAAAATCATGAATCTAACCCCAGAAAAAAAAGCATTGAG 9601 ATTCTGGGACACTCGGGTGGTAGAGAAGATCATCTGATCCTGTCACCTTTCGGGTACGTC 9661 ACTTTATTAATCTCTCTGAGATTCAGTTTCATCACCTCTGAAGTGGTTTGTGTCGACGTA 9721 CAGTCCTCAGGACTAAGTAAGGCCACTTGGTGGCTGTGCCAAAGCACTGTGTCAGGGACA 9781 CGGCAGATGTCTGACACATCTTGTTAGATTCCTTTTCTGTCCTCCGCTCCCCTACCCCAG 9841 AGGTGGGTACAGCCCCATGGCACCTCATCTTTAATGGCTTGGGTTTCTTTTCTCCAGCCA 9901 GGAAAGTTGTCGCTTTGGTGACAGCTATTTTAAGTCAACTGACCTTTCCTGCAAATGATC 9961 CAGATGCCTCTATCTTAGGCTGGTGATGACGAAGATGGCCTATGACGGGGTTCCTGGGGG 10021 TGTGTTGGGAGGTGGGGCAGGGGTGGGGCCCGGCATTTGTCAGACCCATATGATCTTCTG 10081 GCTCCCGGGCTCTGCAGATTTCTCCTGCTGGAGATGCCTACCTGCCAGCAATCTTGGAGA 10141 AGACAGAAATAGCAGCTTTGGGTTCCAGGTCCCCTCCTCCCTTTGGCCCAATGTAGCTAG 10201 AGCTTTGGTTTCCTGCTGCTGTCTTGGTGCCTGGAGCCCTCTCTGGATGGTCATGGAGTC 10261 TTGTCAGAGAAGCAACTTTGGGCTGGCAGACAGTCATTCCAGAAGACATGATCTGGAAAA 10321 ACTGCTTCATCGTTTCCTTCAGAGGCACTGTCCCGAGCCCATTTCCTTGTCTGGTTCCTG 10381 AAATCTCAGGGATGCCATCAGAAGAAGGTGTTCTTGTGTTTACTTTGGACATGGTTTTCT 10441 GTAGTGCAGACTGCCCTTAAACTCTACGTAGCTGAAAATGACCTTGGTCTCCAGACCTCT 10501 TGATCTGTCAGCATCCCTGGGAAATCCAGGGTTCTGTAATCCTCCCCTCTCACCTTGACT 10561 TACTGTACCAGCATCAAACATCCTAAACAAATCCAGTGTTTAGCCAAATACAGCGGTGCA 10621 TGTCTGTAATCCCAGCCACCTGGGAAGCCGAGGCAGAAGGATTAAGGGAGCTGGAGGCCA 10681 GTCTGTGCAATTTAGCAGGACTGTCTCAAAACAAAATTTAATGGTTAGGGGTGGGCATGT 10741 CATTTATTTGACTCTTATCACATGAACACACCTGTAATCTCATCACGAAACGACAAGGCA 10801 GGAAAATCAAAAGTTCAAAGTCATCTTTGGCTACATAGCAAGTTCTAACCTGACCTAGGG 10861 TATGTAAGACCTTGTCTCAAAAGCAAACAAACAAACCCCAAATAACAACAACAACAAAAC 10921 AAAAAGCAAACAAGGAGAGGGTGTGCAGCTAGGGATATAATTCAATGGGTGAGGGCTTAC 10981 CTCACATGCACGAGGCCTTGGTTTCAACTTCCAGTTGAAATGAAGTTTAGTGGTAGAGTT 11041 CTGTGCAAGGCTGTAGTTTCAGCTCTCCATACTGCAAACTGGAAAGAACAACAGTGACAA 11101 ACAGAAACAAAAAACCCCCACAAACAATGTGCTTTCTCACTCAATAAAACCACCTCTTTA 11161 CATACAACTACAACTGCTAAGAAAGTTCTTCAGTGTTCTAGAGCCTGAGCACCTCAAATG 11221 GTTTCCATAAAGCTGTATGCAAACACTGATAAGCCACGAGAAGCAACTGTACAAAGCACC 11281 CTTTGATTTTCATAGTTTATCTACACAAGGATTCTAGGAAAGTGTGCTAGGAAAATTTTA 11341 TGTATCAGCCTTGCGGGTTTGTCCAATAGTTTTAGATTTTGCCAGTGAAGATTTTCCTTT 11401 CTTTATTTTTTACATGGGAAGGAAGTTTAATTGGGGGAAGGGACGGGAGTGGGCTTTATT 11461 TTTATTTTTTAATGAGACTAGCATTTGCATTGGTGGACATTGAAGGAAACAGTTTCCCCT 11521 CCCTAATGTGTGTGGGCCTCACCTAACTCATTGAAAGTCTTAGATAAAACTAAGCTGAGT 11581 GAGTGAGTTGGCCCATACCTGTAGATGGAAGGAAAAGGGTCTTGAGTTTTGGTTTATCCT 11641 AGAGAGAACTTGATCCCCCAAACACCAAACTTTCAAACCAAACCCCAGCCTCCTCAGTGT 11701 GAAGGGATGCTGTTACATGACCACCTATGGACTCAGACAACCTCTCTTCCCTGAGTCTGC 11761 TGGCTTACTCATCAGAGTCTGGGCTCACGAAGCCGCCACACATATATGAGCCTCGTTCTC 11821 CCCACTCTTCTCTTGTGGCACTGAGGTTCAAACCAAGGACCTCGCACATGATAGCAAATA 11881 CTGTACTGAACCATAGAGCCAGCCCTTGTCAGTTTCTTAACACAAACATATAGATGTATA 11941 TGTATATGAATATTTCCATGCTACCAATTCCATTTTCTCAGAGAACCAAAGAATACACCA 12001 AGTAGTCACACTTGAAATTCTGTTCTGAGATTGAATAAAACCTGATCAAATGTGAATTCG 12061 GTCCCTTCTCCCCCATCCCTGACGCCACCACGTTGCTATACAGACCAGGCACAAACTCTT 12121 CTCCTTGTGAATGTGTGTAACACATGTTACCACTGTGCTTGGCTTTTGTAGTTAGAAGGT 12181 TGGTTGATATTTAAAAAAAAACTTTAATATTTAGTCATTACTTTTTAGTAAAGATTTGCC 12241 TTGCTTTTATTTTATTCATGTGCATGTGTGTGTATCTGTGTGAGTGTATGCCACGTGTGT 12301 TTGGGTGCCTCTGGAGATTGGAAAAGAATGTCAAAATCCCAGGACCTGGAGTTCCAGGCA 12361 GTTGTAAACTTCCCAATGTGGGTAATTATAATGAACTTGGATCCTCTAAAAGAGCAGAAC 12421 TCACTCTTAACTGATGAGTTATCCTTCTACCCCCAAATTTATTTGTTTTGTTTATTTGTT 12481 TATTTATTTGAGAGGGTCTCACTGTGTAGCTCTGACAGTATTAGAATTTACTATGTAGAC 12541 CAGACTTGATAAATGTCTAACCCTAGAAAAAAATAGTTTTGTTTTGATTTTATGTCTGTG 12601 CCATCCACTCCTTGAACATATATTTGGTATCTGTGAAGCCAGTGAAGGCTGTTGGTTCCC 12661 TTAGGACTGGAGTTACAGATGGCTCTGAGCTACCATGTGCATGCTGGGAAACAAACTCAG 12721 GTCCTTTGGAAGAGCAAAAAATGTCCTTTGATGGTGGTGGTTTGAATGAGAATTGCCCTA 12781 TCGAGCATAAAAACTTGGCAGCTTTGGCTACATGGTTCTGGATTAAGAGTCAAGAAGGAT 12841 ACAAGAAAGCGGTTGTGGAATCATCCCCCATGGTTAAGGAAAACCACCAAAGCCAGGCTT 12901 GTGGCAGGGGAGTTCCTGCATGGAGGCCAAGAGAAGCCACTATGTCAAGCTGTGAAGGTG 12961 AAGCCTGGATTGTGTTGGAGACCCAAGCTACTGGAGATGTAAGAGATGTGAGATAATGCC 13021 CAGGAGAGCTGCAGACAGGGCATGGAATCAGGCCAAGCGAGAGAAGTGTGTTGCAGTCAG 13081 CAGAACTGGGAGGGAAGAGTCATCTAAGTCCTTTGTCATCAGACATAGAGATACAGGATC 13141 TGAAATTTGCTCTGCTGGGTTTTGGTCTTGATTTGGCCCAGTACTTCCTAACTATGTCCC 13201 CTTTTCTCCCTTTTAGAATACTAATTTATATTCTGTGCCATTGCCGGTGGATCAGGATGG 13261 TTCTCAGATACTGTTTTAGTTCCATGCCTGTCTACTTCCCGTCATGACAGTCATGCACTA 13321 ACACTCTAAAACTGTAAGCAAGCTCCCAATGAAATGTTTTCATTTATAGAGGTGCCTTGA 13381 TCATGCTGTCTCTTCACAGCAATACAACAGTGATTAAGTCAGCTGCTGAGCAATCTCTCT 13441 GGCCCCAGAAGTATGCATGTGTGCAATTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT 13501 GTGTGTGNNNNNNNNNNNNNNNNNNNNNNNNAGGAAATGTCATTCTGTAAATATGTTTAT 13561 CTTATTGGTTGATGAATAAAACACTGTTGGCCAATAGGGCAACAAAATAGGTGGGGCCAG 13621 GATATAAGGAGGATTTTGGGAAGTGTAGGCAGAGGGGAATTGTCATATGATCCCAGGAAG 13681 AGACATAGATGGGCAGAAACTGCCTCTAGCTAACCATAGAGGTCTGGAGGTCTGTACAGA 13741 CAGGCAGGAAGTGATGTAGCTGGAAGAATCAGAATATAAGCAGGAACAAACAGGAAATCG 13801 AGCTCTTCTTCTCTCTCCACTTCAGAGATGCTGAACAGTTGAGATGCAGGATGCCAGAAG 13861 AGTAAGAGGTCCCTGGACCTTTCTCCAGTAAGATAAGACCATGTGGAAATAGATTGATAG 13921 AAATGGGTTAGAGATTAAGTCAGAGCTAGCCAATAAGAAGCCGTAGATATTGGCCAACCG 13981 TTTCATAATTAATATAGCATCTGTGTATTTATTTGGGGGACCTGGTAGACCAGAAAACTC 14041 GTGTTAGAGACATCTTATCAAAGTTGAAAAAAGAAAAAATGTGATAAAGTTAGGAAAAAA 14101 TATAGTAAATGTTAAAAGCTAAATTCTAAAACTACAACTTATTTATCATTTCCTAAATGT 14161 TTAAAAATATTATTTTATAATGAAGATACTTAAAATTCATTTCTCTGTCTTTTGAGACAG 14221 GGTCTCAGTGTCCTGGAACTCATTATATACAGCAGGCTGGCTTGGAACTCACAGAGATCC 14281 ACCTGCCTCTGTCTCCTAAATGCTGGGATTAAAGGTGTGTGCCACCAAGCCTCAATTAAA 14341 ATGCGTTTCTTTTTCTTTCTTTCTTCCTGTCTTTCATTTTTTTGTTTGTTTAGATTTTTT 14401 TTTTTAGACAGGGTTTCTCTGTTAGCATTAGTTGTACTGGAACTCACTCTGTAGACCAGG 14461 CTGGCCATGAACTGAGAGATCTGCCTGCCTCTGCCTTCTGAGTGCTAGGATTAAAGGCAT 14521 GCACCACCACTGCCAGGCTTAAAATGTATTTCTTTTTTTAATTTAGAAATTTATTCTGTT 14581 TAATCCACACGCTTTATATAGCTTTAGTTAAGAAATAAAATAAAATGAAACAGTGAAACC 14641 AAGAGACTATGTCCAAGTCCAGGTCCTCCCAGCCTGCCAATGCCAAGAGCTCTTTAGTTC 14701 TGTGTACCAATTGGAAGAGTAAGAAAAAAATATGGATGGGAACCACACAGTTTCATAAAA 14761 CAGATTTATGGAACTGAAGGGTCCTTGCTGAGTCTAGCAAATTGCCTTTACAAAAGAGAA 14821 AGAAAAAAGGGGGAGGTAGAAAAACAAAACAAATCAACCCAAAGAGGACAAAATCCCAGA 14881 GTTCTAAATTGACTTAGGAACCTGTCACACTGGGACAGAAGCTTCAGCATCCATGAGCTG 14941 TGCCTCCCCTGCTCTCTAGAGCTGGGATCTCGAGGTGTCAGCAGAGACCCCACAGGTAAC 15001 AGGAGCAAAAACACTCACTCAGACCTTTGTGGTACTTCAACAGTGGTCTCACTTCTGGGC 15061 AAGCTTACAAACCTATACAAAGTTGAAGGTGTACTTTACATGAGTGCTAAACTTCAAGAG 15121 GAAGGAAGAAAAAAAGGGAGGTGGAGGGGACAGAGAGAGAGAGAAAAAAACAAAACAAAA 15181 CAAAAACAACCACCTCAGGAGAGGCAAGGGCATTTAAAGGAACCACAAGAATGCCAACGA 15241 TATTAAAATGTATTTCTTAATAGTAAATTTTATGGGAAAAGAGAGTCTCCTCTTCCTCCA 15301 AGTAGGCTAGGTAAGTACCTTGCCACTGAGCTCTATCTATACCCTTCAAAGTGGACAAAA 15361 TGACAAAGATAGTTCATCTCCCCCAAAGGCCCTGTTGGGGTGCTGATTGTCACATCTGGT 15421 GAGATTTCTGTTTTTGTTTTTATTTCAAGACAGGGCCTCTCTACATAGATAGTCCTGGCT 15481 GCCCTGGAACTCACTCTGTAGACCAGGCTGGCCTGGAACTCATAGACCCACTTGCTTCTG 15541 TCTCCCAAGTGCTGGTGCTAAAGGTGTGCACTGCCACTCTTTTTAAGTAACTATGAGTTT 15601 CAAAACAAATTAAAGAGCACTGTTAAAGTGGCTTGTTGTGTAAGCCTAGCTTCAAGTCAA 15661 AGGCCCGAGGCTCCCCTACCAACCAGCTGCTATCACCTAGACACTGTCTGTAGATCTTGC 15721 ACTGACTCAAAACTGTGGCCTAAGGTCAAAATAATGGTCTTCCTGGATTCTGATGTGAGT 15781 GAGATTGTGTAGGAGGGCTGGCCGCTGGCCTGGCTTGAGTCACTCTCAGCTGGTTTCATC 15841 CCATTCCTGCAACTCTGTGTAAGAGGTGGATGATCCTTGCTTAACTGATGAAGAAACCAA 15901 AGCTGTAGAAAGGATCATTTGCTTAACTCTTCACAGATGGCAAGAGGCAGAGTCAGGATT 15961 GGCAGAGTCACTTCTGCCAACTTCACCCTCCTGCTAACTCCACCCTCCTGCTAACTCCAC 16021 CCTCTTGCTTATACTTGACAGTGGAGGAAAAGCCACTGAGGGAATTAAAAGTTGTTACTG 16081 GTAATGGTCAGGAAAAAAGCTGAACAAAGGAGATTAGATTCAGGGATCTTTTTCTGAAAA 16141 GAAAGAAAGAAAGGGGGACTATAGTCTAGAAATGCTGAGATAAAAGGGTGGATTATCATA 16201 TCTACTCTCAAACTAAAGAAGCAACTACTAGTCTCAAATACTTTATATTGGTATGGATTT 16261 TTGTGTATTGGTACAAATTTAAGGTTATTTTTGTTATACTGTATATATGTTTTTCTTTCT 16321 TGTTTAAGGTATTGTACCTGTATAGCTTATTTAAAAATGCAATGTAAACATATAGTCCTT 16381 GAAAACTATTTAAGATAATAAAGAAATACAGGTTAATAGTCATCTATAGCAATCAAACTT 16441 ATAGTCATGTTAGGTATGTTTTCAAGGGCATACAGAAATAAATTTGAGATAGATAGGTCA 16501 TCTTCAAACACTCCAGAGATCTACAGAAAATGGCATTTATAAAATGTTTTAATGACATAA 16561 GATTTTTCATGATAGTGAGAAATGTCTACTCTTGGCAGCACCAATTTACTTCAAAAATGG 16621 ACAATGGGCATTGAAGAAACTCCATGTGGATTTTGCTTTCTTTGTGGCAAAAATCTAGCT 16681 ATCTGGGCAAGAAACTTCCCTTACCTTGACTGCTGTCCTAACTGGACAAGCAGGACATAA 16741 AAGAAATTGACTGCTGAACTTTGCCAAGATAGTATACATTAGTCTTTCAAAAATCCCTGC 16801 TTTACAAAAAAGTCTATCAGATATTCTAAGCTTCTAGGCCAAAGATGGATGCTTCAATGT 16861 TAACAGAGGAATCTTCTGTGACTGATGTTTCTGTCATTTCTATAGTTTTGAAAATTGCTT 16921 GCTCTGTTCTTCCCTGTTTGCTCAGGTAGTATTATTTCCTTCTTGAGTGTCTAATGGAGT 16981 TAAAGACTAGATAGTTATAGCTACAGTTTTCCTTGTAACCAAATTCAGAAAAGAAACTCC 17041 CAAAAGAGGTGTAAAAGTATGAGGCTGAGAAATATAAAAACTTAAATTTATCTAAGAAAA 17101 TGTTTTGTTATCTAAAAAAAAATAATTTTGGGTTAGTAATACAAGTTAGGATAGAAAATG 17161 AATTAGGTACAAAACTTTGGACTCATCAAGAAAAAATAGATAATGGAGTATTTTCTCTGA 17221 ATTTGCCAAATACAAATAGACTGGGTATTGTAAATGTAATTCTTACTTGATAATTGTTCT 17281 TATTGTTTATAGTTTATTATGTTAGAGTCAAAACCTTTCTTTTTTATTTAGACAAAAAGG 17341 GGGAATGTAGAATATTTCTTTACACTGTGTGAAGATGTATCACTGTGATTGGTTTAATAA 17401 AGAGCTGAATAGCCAATAGTTAGGCAGGAAGAGGTTAGGTGAGACTTCTGGGAACAGAAG 17461 TCTCAGGGAAGGAAACAGGCTAGGTCACCAGCTAAATGAAGAGGAAATAGGACACTCAGG 17521 AGGAGAGGTAACAGCCACAAGCCAAGTGGTGGAATATAGATGAATGGAAATGGGTTAATT 17581 TAAGTCATAGGAGCTAGTTAGAAACAAGCCTGAGCTAAAGCTGAGCTGTCATAACTAAAA 17641 GTGGAGCTTTCATAATTAGTAAGTCTCTGTGTCATGATTTGGGGGCTGACGGCCCAAAAA 17701 AGCCTGCTACCCAAGTTCTTTTCAATTTTCAAGTTCTAGGATTCTGGCCTTTTATTGGAA 17761 AACACTGTCAAGTTTCTATAGAGGTCTGACTCCACAGTGTTGCCTGTGCAATGAAATTTA 17821 TTTAATTTATTCCGAGGCCTTGTGCACTCTGGATAATCACTGTACCACTTAATCTATATT 17881 CCCATCCTTCATTATAATTTAAAATGGTCTTATTAATCTGGTCACTTGGCTTTTTTTTTT 17941 TTTTTTTTCTGAGACAGGATTTCTCTGTGTAGCCTTGGCCATCCTAGAACTTGCTCTGTA 18001 GACCAGCCTGGCCTGGAACTCACAGAGATCCACCTGCCTCCCCTCCAGAGTTCTGGGATT 18061 AAAGGCGTGTGCCACCACCTCCCAGTGAGTTTATGTCTTTGCAAATTATACATGGTTTCA 18121 GTTTTTTTTTCTGTTTGTAAGTCACTTTATTTCAAATGTAAAGTTTAAAACAAGAAGCAA 18181 ATTACTATGAATTTTTGTTAACAGTCATTTTCCTTAACTAATAAGTTTTAAATTTTCATT 18241 AATATGTTTTGATCATATTTTTTCCATGCCCCAACACCTCCAAAATCTCCCCACTCATTC 18301 AGTTCTTTCTCTATCTCAAAAAATGAAAAATCCAAGCAAACAACCATTAGACAAAAAATA 18361 ACAAAACAAAACAAAGCAAAGCAAAATAAAAGCACACGGGCTGGAGAGATGGCTCAGAGG 18421 TTAAGAGCACCGACTGCTCTTCCAGAGGTCCTGAGTTCAATTCCCAGCAACCACATGGTG 18481 GCTCACAACCATCTGTAATGAGATCTGGTGCCCTCTTCTGGTGTACAGATATACATGGAA 18541 GCAGAATGTTGTATACATAATAAATAAATAAAATCTAAAAAAAAAAAAGAAAAAAGCACA 18601 CAAAAAACCCAGAGAGTGTGTATTGAGTTGGTTAACCCCTACTCCTCTGGAGTGTGATTG 18661 ATACAGCCAGTGCCGCTATTGGAGAACACTGATTGTCCCTGTCCTTACAGGTATCAATTG 18721 TGTGTAGCTCCTTGGTTAGGAATGGGGCTTTGTGTGCACTTCCCCTTTCAGCTTTGTAAA 18781 GGGTGTCCGATTGAAGTTCGTATCTTCTGGGAGAGCATAAAATCAAAAAAAGATAAATGG 18841 ACTCCAGTGAAAAAGGAGCAAGCGGCACCTATCTTTAAGGTAGAGAGGCAGAGGAGTGTG 18901 GTGTGGCCTGTCACAAACACCCAATTCCCAATCAGCTGGCGTCTACCAGGCTGCTTTCAC 18961 TTAGATGAACCCTGACCTCCATGTCTCCTTAACATTGCCATTGTTTAACTGTTAGTGAGT 19021 CTGCCCTCTGTTCACTGAAAGACTTTCAGAAGGTGGTGTCGCCTGCCTTTAATCCTAGCA 19081 CTCGGGAGTCAGAAGCAGGTAGATAGAGCTCTGTGAGTTTGAGGCCAGGCTGGTCTGCAG 19141 AGTTCCAGGACAGGCTACAGAGTGAAACCCAGTCTCACAAACACCGCCTCCACCACAAAA 19201 AAAAAAGGAAACAAGATAGAGTGAACAAACCCAGCTACCTAGACATCTATCTGGTAAACT 19261 GACTCATCCCAATCCTCCCTGCCCTCCCAAAGAGCTTGGCTGGCTCACTTCCCCAAATGC 19321 TCTTCCCCTTTAACATTTAACTAGTTCTTGTCTCTTGTATGGTTTCCTTTTAACTGTATC 19381 CACCACCCCTACCTTGACTTTTGTCCTGGTTGGTTTTTAATTGTAAACTTGACACACAAA 19441 GTCACCTGGGAAAAGGGAACCTTAATTGAAGAATTGTCTTAGATTGGCCTGTGGGTGTAT 19501 TTATAGGGCATTGTCTTGATTGCCAATTGATTCGGGGTGGGGAGTGGGAGGGTAGGGTGG 19561 GGGTGGGAGCAGCCCACTATGGGACTCACTTTCCCTAGGCAGATGGCTATATTAGAAAGG 19621 TAGCTGAGCCTAAGCCAGCGGGTGAGCCGAGCCAGCAAGTAGCATTCTTCTATGGTTTCT 19681 TTCTTTCTTTTTCTTTTTCTTTTTCTTTTTCTTTTTCTTTTTCTTTTTCTCTTTCTTTTC 19741 TTTTCTTTTTTTTTTTTTCTTCCCGAGACAGGGTTTCTTTGTGTAGCTTTGGAGCCTATC 19801 CTGGCACTCGCTCTGGAGACCAGGCTGGCCTCAAACTCACAGAGATCCTCCTGCCTCTGC 19861 CTCCCGAGTGCTGGGATTAAAGGCATGCGTCACCAACGCCCAGCTCTTCTGTGGTTTCTG 19921 CTTCAGATTTCTGCTTTGAGTTCCTGTCTGACTTCCCTCAATAATTGTTTGTAACCTAGG 19981 AGTGTAAGACAAATGAACCCTTTCATCCCCAAGTAGCTATGGATTTAGAGTGGTTTATCA 20041 CAGCCACAGAGTGAAACCAGAACAACTTTCTAGTAGCCTCTTGTTCTACTCCAGCTGCTC 20101 CTCTGACTATTCCTAAAAGGTAGTTGGGCTCAGGGAACCACATCCCGAGAGATTCAGCCC 20161 ATATGAAAATAGCTCCATTGTGTTGAAGAAATGTGACCCTCCAGGATTTCAGGCATCAGG 20221 ATTCCATGTTGAAAATGAAAACAATTATTTTCCTCTCTCTCAAGATTCCTTTAGTCACCT 20281 TCCCTTACCCCAGTTCCTGGCTTTCCTTCTAAACAAATGTTCAGGGAGGTTCAAACAAAC 20341 AGCTGTGAAGAGCAGCATCCCATACCCCCACCTTCCGACCCAACACTTGCCAGTGCTATA 20401 AGTAGACTGGGATCATCCCTGGACACTGTGTTAAATTACCCATGACCAACCTTCTAGCAA 20461 GCTCTCCTTTTCAGGATTTTGTTGTTTGTTTGGGTTTGTTTGTTTGTGACTTGATCTCAT 20521 GTAAGCTGACCTGGAATTTGCTTAATAGCCAAGGATAGACTTACAACCTGTGATGCTCCA 20581 GCCTCTGACTCCTGAGTACCAGGGATTACACATGTGTGGCATCACAATGAAAGATTTTAG 20641 TTTGCTGAGAGAAAAAGTTTTTAAAGATTTTAGTTCACAGAGAGAATAAGTTTCCCACAG 20701 GCCTTGGTCCAGGACAAGGAAGTTGGTCCCAACCCGAGGGCAGACAAACAATCCTTTTTG 20761 GGTCACACCTGGCTGGCCAACAGACAATAAAGGACTTCTCAGGGTACATTCTATGGTTGA 20821 CCACTCTAACATGAGATCATACTTTGTAATCAATCACTTTGTGCCCCTTGCCTGTATGCT 20881 GATCTGCGGTTTTTTACAGGCTCCTATATAAGGAGTCTGTAACCCTTGCTGGGGTGTGCA 20941 GCTTCCCCGATATTGCTGACACCCGAATGAGCATTCGTTCAATAAACCCTCTTGCTTTTG 21001 CAGCTCTTGGTCTGGTTTCTGAGTCTTGGGGCCTCCTTGGGATCCTGAGACCCTTAAGGG 21061 TCTGGGGGTCTTTCAACACTTAACTTTCCTGTTTTTAAGTAGGAAGATCTGAAATCCCAG 21121 ATTCCTGACTCCATTGCACATTTTCTGTATTAGAGGCTGTAGCTCTGTATAGTGGGTTGT 21181 GTGGCTTACACATGCTCTGAGCTGGAGATTCTAGGGACACTTAGGGTAAAGTGGAGTGTC 21241 AGCCCCTTTCCCTGCTAGACTGAGGCCTTTCTGTTCTTTCCTAACTGGGAGGCTGTATAG 21301 CACCCAATGTGTTCATTAAACTCCATATGTTAGCACTGCATGGAATCTGACACACACACA 21361 CACACACACACACCCTCTACCACCACCATCATCAGCACCACCCCCATCAGCACCACCCTC 21421 ATCCCCCCACCCCCCACCCTGCCCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNC 21481 AACTGGAGGGTAGCATTAGCACCCAGATGCCATTAATGTGCCAAATATTTGCTTGCTTGC 21541 TTGCTTGTTTGTTCCAGCATCCTTAGTGAATGCTCCTGCCCTCCTGGTTAAAGATGGCTT 21601 TGGCATCTCTTGGCATCTTTCTTGTATTCTAGGCCTGAAATAGGGATGAATGGTGAAGGG 21661 CAAGGAGCTCAAGTGTCACTTACCACCTGCACTTGTCCCTTTAAGGGGTTTCCCTAGAAG 21721 CAGTCTACATTTCATTAGCCAGAGCTTTGTCACCTGGCTACTTGTGAAGGAGGTGGTGAA 21781 GAAGCCTTACCTTTGACTCTGCCACTTGGAGCCAAGTCAGGATTCTCTCCCTGGAAAGGA 21841 AATGGAAGATTAATACCTTGTTGGTTGTTAGACCTAGCCCATTATGCGCCATGAGGAAAG 21901 AGAGACAACAGTGGGTCACTGATTGATCAGGGTTACAGGACAAGGAGCCTTGTTTCTCCT 21961 AACAGCTCTGAGCGGAGACAGAAGTGGAGTATATAGGCATAAAATTCACAAACATTTGCT 22021 GCCACGTTACAGGTACATTTTTTCACCAGTCAGAAATCAAAGATTAGGGACTTTGCTTGT 22081 GTGTTCCATCACTGTCAACTGACATACACGGCAAGCCTTTTAGTCCAACCAATCAGAATC 22141 ATTTGTTCCTTCTGTTGTTAGGAGCAGCCATAATGATTCTAAAGAACTAACAATGCATAA 22201 TGACTATTTTTGTAGTTTAGGGATGAGGTATGTCAGCCATTGGACAGTTCTCAGCTCCCC 22261 TAGGGCTTGGGAACTTGAACTTTATTTCATCCTGCATGTAATGGAGTCTGAAGTCAAAAT 22321 GGCAGTACTTAGGTCAAGGTGCTCGTGCCTGCTGCCTTCAAGGTGGTTTCCCATTCCCAC 22381 CATACCAGAGACTTCCTACTGCATCTCCAGTCAAGGACACAAACACTTTTAAGTCCTGAC 22441 TGTTGATTCAATCTATATAGTTACCAGCATAGAGGCTAAGAGTCACACTGGCTTGCAGGG 22501 GACTTCTCTAGCATATGTGAAGCCCCGTTTGAATCCTAAACACAAGAGTCTAAGCTTTGG 22561 AGTCAGAGACAAGCATGTTCAAATCTGTACGTCACCACCCTATAGACATAGACAAGTCCC 22621 TTGGGCTCAGTTTTTTCACTACAGAGAGTAATTGTTATTTCAGATTCCTAGGGTTGTGGT 22681 AATTAAATAGTTGAAAGATATAGCCCATGGAACATAAAAAAAACTCAAAACCAGGCACAG 22741 TGGCACATGTCTTTAATTTCAGCACTCAAGAGACAGAGGCAAGTGGATCTCTGTGAGTTT 22801 GAGGCCAGGCTGGTCTATATAGAGAGTTCCAGGTCTACACAGAGAAACAGGCTCAAAACC 22861 AAAGCAAAAGCAAAACCTCAACTAATGTTCATAAAATTATGAAATTGCTGGTACCAGTGA 22921 CATGACTCATTGGTAAAGACACTTGCTAGCAAGTTTAATGATCTGAGTTTTATCTCCGGG 22981 ATCTACAATGTAGAAGAAGAAAAACAACTCTCAAGAGTTGTCCTCTGATTTCCACTTATG 23041 CAAAATAGCATGGGAACACACTTAAGCAGGTAGGTAGGTAGGTAGATAGATAGATAGATA 23101 GATAGATAGATAGATAGATAATAGACATAATTAAGAACGTTCAGTTGCAGCACAGTTCAT 23161 ACTGAACTGCATTTGGACACCTCTGTGAAAAGTCAGGAGCTCTCCTGTCCTCCTGGTGAC 23221 ATTTAAACATTGAAGGCAACTATTTTAACTGTCAGTTATATACAAATCCACTGGCCTTGT 23281 AAAATTTTAAAACATAACAGAGGAGGCTAAAGTCCTGTTTAACAACCCTCTCCTTTTACC 23341 ATCCCAGGAAGCCAAAATTGTTCACAATTTGTTCTCTTCCCTCAGGCCTTCCATATTTCA 23401 AATACCACATAAAACACCTATGGAAAAACATGAGGTATTAAAAATGTCACTTGGAAATCC 23461 TTCTTCAAACAAGCTTGTTCTTTCTTTTTTCTTTTATGTACAGTGAATGGAATCCAGGAC 23521 CTTTGCAGATGCTAGGCGAGTCCTTTACCTCATTCCTCTTTCGATTTAAAACTTTTTCTT 23581 GTTTTGTGGAGACAGGGTTTCTCTGTGTAGCCATAGATGTCCTAGAACTAGCTCTGTAGA 23641 CTAGGCTGGTCTCAAATTCAGAAGCCAGTCTGCCTCTGCCTCGGGAGCGCTAGGATTAAA 23701 GGTGTGGGCAGAGTGCTAGGATGAAAGGTATGCACACCACCACTCCTGGTTGATTTTAAA 23761 AAGATGCTTTTTAAAAAAAATGATGTGTAGGTAGTGGGGGGAGAGACGGTTTCATGCCTA 23821 AGAGCACTGACAGCTCTTCTAGAGGACTCAGGTTCAATTCCCAGCACCCACATGGCAGCT 23881 CATAACCATCTGTAACCCCGGTCCCAGGGAATCCAACACCCTCTTCTGGTCTCTGTGAAT 23941 GACAGATATGCATGGGATATACAAACATATACGCAGACAAAACACTGTATACATTAAATA 24001 AGTACAAATTTAAAATATGTGTAGGCATGTATGTCTGCATGTGGGTATGTGTACACTGAA 24061 TGCAAGTTCACTTGGAGGCCAGAGATATATAGATCCCCTGGAGTTGCAGTTACAGATACT 24121 TGCGAGCTGCTGTGAGTGTGCTGGGAACCAAATCCTCTGGAACAGCAGCAAGTGCTCTCA 24181 CCTGCTGAGCCATTTCTTCACCCGCTTCTTTCTACTTTTTATTTTGAGACAAGGTCTTAC 24241 TAAGTTATATATTCACTTGGGGCTTGAATTCATTTTGTCAGCAGGCAGACCATAAACTTG 24301 CCTTCCTCTTGCCTCGGGCTCCTGAGTAGCTGAGACTTCACCATGAGGTCTGGCTTTGAT 24361 TACATTTTTCTTTGTTTTCTTTTTGGGGGTGGGGCTGATCATGAACTCTAAATAGCCAAG 24421 GATTGATAGTGAAGTCCAGATTCCCCCACCTATCACCGGGTGGAATTACAGGTGTGCACT 24481 ACCACACCCAATTTGGTTTGATTTTTTTTTTTTTTTTCAGGACAAGCTCTCCTTTTATAG 24541 CTCTGACTGGGTTGGAATTTACTATGTAGACTAGGCTAGTGTCAAAATCACAGAGATCTT 24601 CCTGTCCCTGCTTCCTGAGTACTGGGATTAAAGGCATGTACCACCACACCTTCGGGTGTG 24661 GTGATGCACAGCTTTAATCCCAGCACTCAGGCAGGCGAATCTCTCTGAGTTTGAGGCTAG 24721 CCTAGTCTTCAGAGTGAGTTCCAGAACAGCCAAGGCTACACAGAGACACTTTGTTTCGAA 24781 AAACAAACAAAAACAAAAGAGGCTAGCCTGAAACTCCTGATTCTACCAGCACCTCCCAAG 24841 GGCTGGGATGACAGGTTGTGGCCCCATGCTCTCTGCCGGGGCCTCTCTTTTCTTTCTTCT 24901 GTTTGAGGTAGAGGCTTACTAGGTTGGCTGGGTGAGTTGTGAACTCACTCTGCAGCCCAC 24961 ACAGGAACTGATCTTGTGATCCTCCTGCCTCAGTCTCCCTAGCAGCTAGGATTGCAGGCC 25021 TGCACCATCAGGCCCATCGTACACTGTTTTCTGAGTTTGAAAATTGCCTCTGTTGTTGAC 25081 TATAAGGCATGCTCTCCTCCTAACATTGTCCTTGGTGCCTCTGCCACCCTTTGGGACTAG 25141 AGAGAACAGATCTTATTCCTATTTCACATGCTGTGCCAACCCAGTAACAAACTCAGATTC 25201 CTGCTTCCGCCCCCACCACCCCCATCTAATTGTTCAGTGTTTCTGTGAAGATAAACACGA 25261 TCATCTTTGTGAAAGCCACTTAAGTTCCTTTCAAGGTTGGGATATAAGTTAGAGTGATAG 25321 CTTGTTCCCAGGGTGGGGAGAGCATGTGAATTCCCCTCTCGCTCAAGTAGGCTATACTAA 25381 TTTTCATTTAGATATTTCTGAGGCAAAGTCTCATGCTGGCCATCCACCTGCCTTAGCTTC 25441 TCAAGTGCTTGGATTACAGGCATGAGCTACAATATCTGGCTTAGTTTCAAGGTTGTGAAA 25501 ATTATACTGTGTTCTGATGACCTGAGTTCAATTCCCTGGACCTGGGTGATGGACGGAGAG 25561 GACAGACCCCTGCAGATTGTCCTTTGACCTCCCTGTCACTATGTGAACACTCGTGTACAC 25621 ACACACACACACACACACACACACACACTAAATGAATGTAATAAAATATAAAAAGGTGTT 25681 CACTAGTTAATAAGACATGAGAGAAAAAGCTTACCATCCCTAATCAATGGGGAAGCATTG 25741 AATATAAGTGACTGTGGTCATGGAAAGCAGTATAGAGGTTCCTCAATAAACTGGAATATA 25801 GCAGCATATACTTGTAAGCCTCCCACAACAGGAGAAAGGTAAAGAGGGGCGGCCACTCTG 25861 GAATATTATTAATATCCTGTTTCATAAACAAGTAAATAGAACAAACCCCTCAACAACAAG 25921 AACCGGTGTGCTGGCACACACCTGCAATCCCAGCATTTGGGACTTGGAGGCAGCACAATT 25981 GAAGTTCGTTCTTGGTCATCCTCAGCTATGTATGAAATCTGAAGCCTGCCTGGCCTACAG 26041 GAGACCCTGTCTCAAAAAAATAAACTAAATAGATTAAAATGAAAATTAGAAGCAGGTAGT 26101 GTGGAAGTTGAATAAGAATAGCCGCCATGGGCTCATGTATTTGAATGTTTAGTGGCACAA 26161 CTTGAGTGAGTTAGGAGGTGTGGCCTGTTGGAGTTGTGTGTCACTGGGAGTGAGCTTTGG 26221 GATTTTAGAAGCCCAAGCCAGGCCCAGGGACTTGCTCTCTTCCTGCGATCTGAGGAACTG 26281 GATGTAGAACGCTTAGCTACTTCTTCAGCACCATGTCTGCCTGCATGCTGCCATGTTCCC 26341 TGTCAAAATGATAATGGACTGACCCTCTGAAACTTGGTCTCTTTTGGCTGAGGAGTTAGC 26401 AAGGTAAGAGGTGGCTGTGGCTTGCTCTTGTTTCTCTCTCTCTGATCTTTCATCATTTTC 26461 TCCCGTATCTGGCTGTGGGTTTTTATTATTAAGAGTAATTAGAACTCATGTTACAGTGGT 26521 ACATGCATGCCACAGACCCAGTGTGGATGCCAGAGGACAACATGTGTAAATTTTTTCTTT 26581 CCTTGTATGTGCGTCCAGGCTAGTTTCAGACTTGTGGGCTTCTGCTTCAGCCTCCCAAAG 26641 GTGGGGACCACAGGCTTATATACCTACACTCACCTCTTTATTCCCAGTGGATGTGTGTGT 26701 GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGTGTGTTTTACACAGACCTGTACC 26761 ACATTCATTTGGTTACTTTTTTTTCCTGCATTTTGTTTTTAGGTAGGGTCTCACTATGTA 26821 ACCCTGACTGTCCTGGAACATGCTATTTAGATTAGACTGACCTGCTGGTCCCTACCTTCC 26881 GAGTGCTGGGATTAAAGGTGTGTACTACCATACCTGGTGATTAGTTTGTCTTTTGAGACT 26941 GGGTCTCTTGTAGCCCAGGTTGGTCTTGAACTCCTGGTTTTCCAGACTCTACCTTCCAAA 27001 TATTGATATTGCAGGTGGTCACTACCATGTGTGGAATTTATTTTTGAGCAGTGTTCTGTG 27061 GGTGGATGATAAGGTCATGTCTATGGTAAAATTGTTTCTAATAATGATGAATAGCTTCAT 27121 GTGTGTATGCATCTATCAGGTTTGTTCAACCTGAAGTGTAGGCCTAATATTTGGATTTAT 27181 TTAGCCAGTGATAGCTATGAATTGAGCCCAGAAAAAATCATAAACTTGACTAAAACATCT 27241 TAAGAATTTTGTAACTTCTTTTGTAACTCAACTGTATTGTTTCTGAGCATGAATGTTGTA 27301 AATGACAATGTCAGCTGCCATGTCAAAAGGTTGAACATTACTTGGCAGTGGTGGCACACA 27361 CCTTTAACTCCAACACTCAGGAGGCAGAGGCAGGCAGATCTCTGAGTTAGAGGCCAGCCT 27421 GGTCCACATAGGGAGTTCCACACCAGCTAAGGTGACAGAGTGAGACCTTGTCTAATTTTT 27481 TTTTAAGGTTGGACATGTATAATTCCAGAGAATAATTTTTCACTAATCGGAAAAGAGGCA 27541 GTTTCAACTTGGAGTTCACAAGATTTAATCTTTCTTTGAAGATTTATTTATTTTTAGTTA 27601 TGTGTGTGTATATATGTATGTATGTATGTATGTATTGGTGTGTTAAACCCCTGGGGCTGG 27661 AATTACAGGTGGTTGTGAACCTGATGTTGTAATAAGCTCCCAGACCGTAGCACAAATGAC 27721 TCTATGAAGAAAGTACCATTCAGGCTGTAAAATCCACATAGACAGCACCACCTGGAAAAA 27781 CTAAAACAAAAATCCAATCCATCAAACTCCACAGATCTGGGAAAGTATCTAAATGCACTA 27841 ACCTTGATTTTTGGCTTCTGTAGTTCTGCTTCTGGCTAACTATTCTTGTTAACTGAAGTA 27901 TGTGAACCCACAACATGGTTTTTGTGCTTAAAAGTTCTCTGTTCTACAGAATGAATTCCA 27961 GGACAGCCAGAGCTGCATGGAGAAAATCTGCCTCAAAACAAAACAAACAAATAAAAACCT 28021 TGAGAAAGGCTCAGGGCTATACTGGTATCCCATACACTCAGTGTAGTCGCCAACTGTCAA 28081 AGACTTTTTGTTGACTTAAACCCATTTCTAAGCAGTATTCTCTTATGGATACCCCTTACA 28141 AGTGGGTGCTGGGACTTGAACTCAGGTCCTCTGGAAAAGCAGAGGATTTCTCACCTGCTG 28201 AGCACCTCTCCAGGCCCATAAGATCTATCTTAAGACAAGACCTGAGCAGCCTTATGGAGA 28261 TGGCAGTCTGGGGAACCACTGGTGCGCCTTTTCTTCTGCTGGTCACAAACTGCTGTGGGA 28321 ATTTCCATCTGAAGTTCCTGCCTCTTCTCACATTCCATGATATGAGAAAGCTATCAATGT 28381 TCTAAATCTGTTTGCTTTCTGCTTTGCAAGACCTTTCTCTTTCCTAGGTCACCCTCCAAG 28441 AGTTCTTGACCTCAGCCCCGACTGGTGTCTTGGGATGGGTGACTGGGTTCTGGGGGCTTC 28501 CCTGTGCCTTGGAATATGGTAAAAGAGCATCTCAGGTATTCACTCAGTAGATGCTAGTAG 28561 CACTCCCTCCCTCCATTTCTGTCTACAGATGTTGCTAGCTGGCCCCTATGAGGTAGTCTT 28621 TGCCCCTTTGTTATTGCTGCAGACTCAGAAAAAAGAGGAAATATAGAACTCCTCGTGGTC 28681 TTCTACTCAATATCCAAGCAAGGGGGAACAACTGAGCATCCATACACTGCTGTTTTGGCT 28741 TCTCAATTGCTTGCTTGTACATCACCAAGAAGCTTTCATTGGTCAGTGTAAACAAGATCT 28801 GGGAGTTGATGGTAGAGCAGTTGGATGAGTGACTCTGTCTTTCACCTTTGTTGAGTCATT 28861 TGGTGTGTGCACATTGTGGGTCCCTGCCTCGCTTCCCATTAAATGTCAAGGTGAACTTTA 28921 TGAGGTTGAAACTTTTATATGTAGTGCAACTGTACTCCTTCCTCTCTATCTCTTCCTTCA 28981 TTTTTCTTCCTTCACCTTCTCTTCCTTTAAAAAAAGAAAAACTTTAAAAAATGTGAATCT 29041 GATGTATCCCAGGATGGCCTCAAACTGTTTGCTTTCTCAGAAGATGACCTTGAACTTTCA 29101 ATCCTCCTGCCTCCACCTCCCAAATGCTGGGCTTACAGGAATTCATCACCATGCCTGGTT 29161 TTCCTCTCTCCTGGTGAGTGAATCCAGGGCTTCATGCTTGCCAGGCAAGTGTTCTGCTGA 29221 CTGAGTTACATGCTTAGCCTGTATCCACATCTTGACTGAGTAATTTCTGCACCAAAACTT 29281 TAGGTTTCATCTCAGTGACTCTGCCAATGTGTTTCCATTTTAGAGTGACGACTGGCCTTA 29341 GAGGAGAGTGTAAGAGAAATAGAGTCTCTTTCCTTGGTCTGCTTTTTAAATTTTAATTTC 29401 TTTTTAGACATCTTATATTTATTCATGCATGTGTGTGTATAACTAGCAGAACTCAGCTGT 29461 CTCTTTCTACCACTCAGGTCACCAGGCTTGGTGGCAGGGACTCTTACCTGCCTTCGAGCA 29521 GGCTCTGCCCTCCTTTTGGAGAAACTGGTTTGCAGAAGGAAGAGACAGCACAGCTCAGAA 29581 GACAGCCGTGCTTTCAGATGCCTGAGAATCCTGCCAAGGACACTGCTGCATTCTCCTATT 29641 CTTTTGTAAGGGTCCCATCTCTGCTGAGCTAAACTGGGCTTTCTCAGCCCTTCTCCTCTG 29701 ACAGTATTTTAAAACCCTACCTAAAGGGGGATGGAGAGATGGCTCAGCAATTAGGAGCAT 29761 ATCCTACTCTTCCGGAGACCCCTACTTCTGTTCCCAGCACCAATGCTGGTCAATTTACAA 29821 CTGTAACTCTGCTCCAGGTCATCGGATGCTGCTATCCTCCTCAGGCAACTTCACTCATGT 29881 GCACATACACATACTTAAAAACAAAATAAGTCTTTAAAAATCACCTAAGAAATATAAAGG 29941 CACATATCATAATTCAGCCTGCTGTGACGTATAGCTATAGTCCCAGAATTCTGAAGGCAG 30001 AGGCAAGAGGATCACCTCAAGCTTGGGGCCAGCGTGGTCTACAGTGAGACCCTGGAGACT 30061 TTAATCTCAAAATATGTAACAAAACAAATATGTAAATAGACATATATCACAATTTATATT 30121 TAAGTAAAATGGGGGGCATTGGAGAGATAGCTTTGTGGTTAAGAGCATGTACTGTTCTTG 30181 TCAAGGACCCAAGTTTGATTCCCAGTGTCTACACTGGTTGGTCTCCAACCCAATTCCAAG 30241 AGATCTGCTGCCTTCTTCTCCTCTCTACTGGAACTGCATTCATGTGCAAATGTCCATATG 30301 CACACACATACCCACATGCATACACACAAACACATACATACTCATTTTGCCTGACATCGT 30361 GGTAAAGTGGGAAGACTTGTTGCCCTATTACTTGGTCTTCATTTGCCTATGAGCACCATG 30421 TTGGCATGAACTCATTCATTAATATCTTTCCTGTACAACTCCCCAATAACCAAGATGACA 30481 CTTGGCACACATTAATTGCTAAGTATAATGAAAATTTAGTTTAAATTAGCTAAATAATTT 30541 AAAGTTCCCCCTCAAGCCTCATGCCTGATTTAAAGTAGTACTTATTAATGCTGGGCCTGG 30601 TGGCATACATTTCTAATTCTAACACTTAGGAGGCTGAGGCAGGAGGATGGCCAATTCAAG 30661 GCCAGCTTAGCCAGCTTAGTAAGACCTTGTCTCCAAGCAAATTACAGCAAAGTCTGAGAT 30721 ATAGTTCAGTAATTAGGGTGTTTGTCTACCATGTGTGAAGACCTGAGTTCAGTTTCTAAC 30781 AACAAAACAAAACTAAACAAACCAGAACCTAGAGGTTATCATTTATTTTTTTATTTTTAT 30841 TTTTTTTTGGAGTTTATGCCTTTGGATTATCCATTCTATGTCCAGACATCAGTACTGCCA 30901 TGTTACAGTCAATAAAAGTCTTCCTTCATCACCCTTAATCTTATCACCACTAAAGTCTCT 30961 ACTTGACAGACATGCCATACATAATTATAGCTGTTACCTTCTATCATAAAGTAGACATTT 31021 TATTTTATTTGTGTATTCATTTTCATTTATTTTGTTGTTGTTGTTGTTTTATGAGACAGA 31081 GTTTCTCTGTGCAGCCCTGGTTATCCTGGAACTCACTCTGCAGACCAGGCTGGCTTCAAA 31141 CACACAGAGATCCACCTGCCTCTGCCTCCTGAGTGCTAAGATTAAAGGAGTGTGCTGCCA 31201 TCTTCCCAGCAACATTCTAAATTATTTTTTGTTTATGTTTTGAAATGGTCTAATGTAGCT 31261 GAGGTGGGCCTCAAGCTTGTTATATAGCTGGGGAACCTTGAACTTGTGTTCTTCCTACCT 31321 CTAGAACTCTGGAGTGCTGGAATTACAGGTATGAACCATCACATTCCAGTTTTAATCAAA 31381 TCCAGACTTCATGGGTACTAGGAAAGCACTCTACAAATTAAACTTCACCCCTAGTTCATA 31441 TATATATATGTGTGTGTGTGTCCATGTATGTATGCCTACATGATTTTATGTGTGCCACAT 31501 GTGTGCAGGTGCTCTTGGAGGTCAGAGGGTGTCAAATCCCCTGGCACCTGAGTTATAGGT 31561 GGTTGTGAGCCACCTGATGTGGATTCTGGGAACTGAACTTTGGTCCTCTGCAGGAGAAGT 31621 CACTGTTCCTCTGAGTGAACGTTTCTACTTTTTAATATACTTCCCATTCGAATTAGAAAG 31681 TAGAAGCTCTCGGAGGTTGAGACCTTACCTAAAGTCACCCAACTAGTAAGAAAACTAAAA 31741 TATCAACTTGGTTTTCTGAGTTTTAAATATTTTTTCCCAATGTGTAATTACACAGGAGAA 31801 TTAATGGGGACACTTCAAGGTAAAACAGAAGCTTTAGACATAGCAAGGCATGGTGGCACA 31861 CATCCCATTGAGAGGCAGGAGGATCAGGAGGCCAGCTTTGGCTGCATACTTAAGAGGCAT 31921 CCAGGGCTACATGAGGCGCTACCTAAAAAAATTAAATTAGGCAGGGCGTTGGTGGCGCAC 31981 GCCTTTAATCCCAGCACTCGGGAGGCAGAGGCAGGCGGATCTCTGTGAGTTCAAGGCTAG 32041 CCTGGTCTTCAGAGCGAGTGCCAGGATAGGCTCCAAAGCTACACAGAGAAACCCTGTCTT 32101 GAAAAACCAAAAAAGCACTGGTCATTGTCATTTTCTTTCCTAACAGGGCACTGGAACCCT 32161 GATGTTGGTTGGCTCCTAGATTTCTTCTCCACAGCAGAGAGTTCTTGCCCTGTTAGAGCC 32221 AGAAGGATGCTCTGGAGAGTCAGTATATAGCAAAGCAGGGTCATCTGGAGTAGTAAAAAC 32281 CCTCTGGCACAGTCAGACCTCATTTCCTCTTGTCCTGTGCTCGTGGCTCTAGCATTATGC 32341 AAGGAGAGGCGCAAACAGCAAACAATTTGGAAGGGCTAGCACTTGAGCAACTCTTTGTAG 32401 CTTCCTCTTCTCTACTCTTTTGCCCCTGGCTTCTACTGGAACAGGTGACTTTCCATTGCA 32461 TTGCATTCTCCAAACTCAGATGATTTTGAGAATGTGGCACTACTAAAAGTCACATGGACA 32521 TACAAGGTACAACTAGAACTATCCCGGGAAACAGTGATACACGATCTAGTTTGAGGCCTT 32581 GAGCCATAGCTTGTCAGAAGCTCAGAAATGATTGAGTCTCTGGGAGCCCTCACCTCAGCA 32641 TCCCTGCTTGCAAAAGGCTTCTTGAAGTAGTAAAAACTGCTGGGACCTTGTCTAGGCTGG 32701 GTAACCTTGCATAATTACTCAACCTTACTGAGCTCAGTCCCCTCCTCTATAAAATAAGTG 32761 CAACAGTATTTACCTTAGTGGCCCACCTGAAAACATCACAGCTGCCATAGCTAGCTCTTG 32821 GCTTTTGTTCTATCTCCTCCTCCCCCTACTTTCTCTTCCCTCCCTCCCTCCCTCCCTCAT 32881 TTTTCTTTATTCCTTTCTTTGTATTTTTTTCTTTTTTCTTCCTCACACCTCTCCTTATTC 32941 CCCACCCTCCTCTCTCTCTCTCCCTTCCCACTTCTCTTTCTTTCATGGCAGGATATCATG 33001 TATCCTAGCTATACTTGAATTCACTATATAGCTGAAGAGGAGCTTCCAGCCCTTTTGCCT 33061 CTGCCTCCCAAGTGCTGAGATTATAGGTGTCCACCTCCACGTCTACTTATGCTTTGCTAA 33121 GGATCAAACCAGGGCTTTGTATGTGCATGCTAGGCAAGAGCCAACTACATCGCCAGACCT 33181 ATATAATACCCCTTTCTCAGCGAAACTGGGGTTGCTGATGGCTGGTGTTGGGGGAAGGCA 33241 CTAAATATTTAGCAGAAGTATAGGAAAACTCTAGAAGTCTAGAGATCCTCAAAGTAAGTT 33301 TGGAGAGCCTTGGCCTTTTCTTAGTTGAAAGTCATGGTGCCTACTCACTTTGACTGCTCA 33361 AGGAATATCCATTCACCACCTGGAAATAAGAAAGGAGGGAGAACCAGCTAGGGATGTGAC 33421 TTAGTAGTAGAGCACTTGTCTAGCATGAGCGTGGTCCTGGGTTCAAGCTCCAGTACAAAG 33481 GCTGGGTGGGGGGGTGGAGAAAGGCTTCTTTCCCATGGCGTTCTAGAGATGGCGGGGAGA 33541 AACCACCAATCCACATCTATCTACAACAGTTCAAGTAGAACTAATCTTGGTGGTATGGCT 33601 ATAGTAGTCCTAATCCCATCTCAGGGATGCTTCTCTTTGCAATTGATACAAAACACATTA 33661 CAGAAAACCACAGTGAATCAAAATGCAGAGTTGTGGTGCCTAGTTCCAATGGATGCATCT 33721 ACAGTACAACTCCCATGCCTAAGGCTCAGGGATCATTGTGGAAGACAAAGATCCTCCCAG 33781 GAGATCAGGGAGTTTGCTGTCTCCTAGGAATTTCAGAAAATACATCTGTAAAGGCTCACC 33841 AACGTGAATTCCTAAACATGAGCTGAACAAGGATGACAATAGACATGCTAACAAGGATGG 33901 GAAAAAGCCCTTGAAGCCTCAGACCTACACAAAGAGCCGCAGTTGATTAAGGAATGCTGA 33961 TTGTGGGAGAAACCATCTTCCCAAATTGTTATCTAATACCACATAGTCAGCCCTGAAAAC 34021 ACACATGCAAATAAGATTATACAAAACAAGGGGGTTGTACATATGTATTTAGGAATATAT 34081 ATATATATATATATATATATATATATATATGTAACAATAATTAATAGAAAAAGAGACCAT 34141 GAATTTGAAAAAGAACAAGGAGGGGTACATGGAAGGGTTTAGGATGCTTTGACCCTTTAA 34201 TATAGTTTCTTGTGTTGTGGTGACCCCAATCATAAAATTATTTTTGTTGCTAGTTCACAA 34261 CTGTAATTTTGCTGCTGTTATGAATTGTAAAGTAAATACCTATGGTTTTTGATGATCTTA 34321 GGCAATCCCTGTTAAACTGTCATTCAGTCCCCAAAGGGGTCAAGACCCACAGGTTGAGAA 34381 CTGCTGATTTAGAGAGAGGAAAGGGAAGGGGGGGTGAAATGCTGTAATTATAATTCCAAA 34441 AAAAAATTTTTAAAAATTTCTTAAAGGAACTGAAGAAAAGAGCTGAACATTCTAAGCTTA 34501 AGGGGGGAAAGGTTCTGGAATGTTACATTTTTCTGGTTTCCTTAGTCTCAGCAACAGGCT 34561 CCCAGCCTTCTGTTTGGACAGTGGTTTACAGGCATGTGAGCTCAGGGAACACTCTTCCAA 34621 GTGAATCAGACTTCAGGAGAAGACATTCAGTTCAGGGCCCTGGGGAAAGTAAGGACAGAA 34681 CTCCATTCCTGAGAATTACCAGGTTTGCTCAGAAGATAAAACTGGTGAGCCCAATGGCTG 34741 TGTGCACAACCCTGACCTCAGTGTCTAGGATAGCTGGACTCTAGCTGCTAGAAGATAGTC 34801 AGAGGGCCATCCTTTCCCTGAGGCTAATCTGTGAATCAAGTAAACTACAGTCAGGAAGGG 34861 AGCTGGAGATGGGGGCCCAGCAAACAGGTCCCCCTTAAAGCCCAGCACATAGGTGGGGAA 34921 CCCAACCTCCCATTTTGTCTTCACCCCACCACCAGGCCTTTACCAAGGCCCGAGGTTGCC 34981 ACTATTTTCAGCTTGCCAGGCTCTTTGCAGTTTTAGGGGGATGAGGAGGAGATGCTCTGA 35041 GGTGCTGGGAGGCACATGGCGGGTGCTATTTATGGCTTGGGCTGAACTCCGATGTCCTAG 35101 AAAGAGTGTTTCTGACACTTTCTGCCTTCTGGGAATCAGGAGACTCATGACAAACACTGC 35161 CTGGCAGTGTTTCTTTCTTGTTCACAGCAAGAAGTGTGCAGTCCATGGCACGAAAGAGGC 35221 CTGAGCAGGGCAAGATGGACACGATGACATCACTGAAGGAGCTTCCCAGGGGCTGTCTTG 35281 ACTGCTTCATTAACTCATTCATGCAGTTTATTCAGCAGCTATGCCTGTCAGACCCCATTC 35341 TGTCTGCACAAGACACATGGCAACAAAGGAGACTTACTATTCCCATCTTCATGGGTTTTA 35401 TGTTCTGGCAAGAGGAAGATAGTAATAATTTTTAAAAAGTAACCAGTCTTGAGAGCATGA 35461 TAAATATGGTTGATAACAATATGCTATATTTTAAAAGTTGTGAGATAGTATACTTTAAGT 35521 GTTCTCAAAACAAAATGATGAATATGGGTGATATAACATGTTAATTGGTTTAATTTAGCC 35581 ATGCCTTTGTGAACATACTGTATCGTGTATCATAATTGTGCATGACTTTATTTATGAGCT 35641 AAATAAATGAATGGAAAAAAAAGTAACCAGTCTTGATGCTTACCTGCCATCCTGGAAGGA 35701 AATGGAAATAGGATCTGCCGCCGCAGCATTGCCCTATGCTCTTATTTCTTCTCTTGAAGA 35761 GGTAGGGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTACTAGAGACTGAGCTA 35821 CCGGCCTCACACATTCTAGGCAAATGCTCTACTTTATATTAAACACTTTATAAAACATTA 35881 AGCCTTTCAGGGTCAGCAAGGTAGCTCAGAGAGTCCGGGCATTTGCTACCAAGCCTGACA 35941 ACCTGAGTTCGATTGATGATCCCCCAGACTCACGTGATAGGAGGAAGCTGACACCTGTGG 36001 GTTGTCCTCTGACTATAGGCATGCACACACATCCCATGAATAAATATTTATACATTTTCA 36061 AATCATACTTATTTTACAATGATTTTTATTTGTTTGCCTGTCTTTCTGTCTGTGTAGAGA 36121 CAAGGTTTCATGCAGCTCAGGTTGGCCTCAAACTCACTCTGTGGCAAGGATGCCTTAACT 36181 TCAGGTCTTCCAGGTCCAGGTAACAAAATGTTCAGGAGGAACCTGGTACCTCATCATAAC 36241 CGGTTCTAGATGGTCTTCCCAGGGCTGCTGTAAGAAAGTGCTACACGACGAGTTATTTCA 36301 AACATTCTCACAGTTCTGGGGATTAGAAGTTTGAAACTAAGGTGCTGAAGAGATTAGTTC 36361 CTTCTGGAAGCTCAGAAGAGCCATCTGGTCCATACTTTTCTCCAGGTTTCTCTTAGTTTT 36421 TGGCAATCCTTGGAATCCCTTGGTTTGTAGATGCAGCTTCCAAAGCTCAAGATCTCTCTC 36481 CAATGCTGTGTGGCATTTCCCCGTGTTTATGAGTGTCTAAATGGCTTTTAAAAACATTTT 36541 TGAGATGTGAAATTCTGGCTGACCCAGAATATATAAACCAGGCTGACCTTTGTCTCCCAG 36601 AGATCTCCCTGCCTCTGCTTCCCAAACCTTTTGATTAAAGGTGTGTGTCAAGTGCCCAGA 36661 CCAAATGCCCTTCTTGTAAGGACAACGGTCATATTGGATTTAGTGTCTAAGTGAGTCCCC 36721 TATGAACTCATCTCGAACTCAGTTTGCATAGAACACTGTACCATGCAAAATAAATGACAC 36781 AGAGACTGATATTGGGGTTCACACTTCAAGCTGAAGGTCAGAAAAGCAAAGCATTGGGCC 36841 ACTAGCTCTTACCACTACCTCAGGCTGAACGGGCTGATCCTGCTGCCTCTCCTCAGCATG 36901 GCTGGAGAATATCTTCATATCCTCATTGTGGCTGGAAAATGAATGCCTGATATGGAGAAC 36961 TTGCTCCTGTTTTATATAACTCCCTAATGCTGGGATTAAAGATGTGTGATCCCAGGTGCT 37021 GAGATCATCTTTGTGTGAGCTGTTTCTCTTTAGGACTGGATCAATTTTGTGTAGATCTGG 37081 ATGGCTTTGGGCTCACTGAGATCTATCTACCTCTTAATCCCTGGTCCTAGGATTAAAGGT 37141 ATGTACCACCACATCCTAGCTTCTGGCTGCTGGGATTAAAGGTGTATGCCTGGCTTCGAT 37201 GGCTTGTGGCTGACTTTGCTTTCTGAATCCGCAGGCAAGCTTAAAAAAATCATAAATAAT 37261 ATATCACCATAGACCACACTTCCAAATAGGCTTCCATTTAGAGGCGCCAGTGGGTGATAA 37321 TGTAGGCGGTTTTACTCAGTTTTGTGCAGATGGCTGGCGTCCTGTCTGGTGAGTTCAGAT 37381 TTTTTTTTTTTTTTTTTTTAAGTTCAGAATCTTACCCAGCTCAGCTTTTCAGGCTGCATT 37441 CAGTGTCCGGCTTTTTTCTCACCGTCTTGACTTCCTGTCCTGCATCCCATTTCTCAGCCT 37501 GGACCCTGCCAGTCTATCAGATAGATAACATAAACAAAATTGTACTGGATTAATGGGAGC 37561 TGTTTGGACATTTCCTACTTTTGCCTTTTCACCAATGATTTGCATACTTAAGCCTGCAAC 37621 TACAGCCCCGATGCAGTAAGCTCAGTCTCTGGCAAGCAAAGGTCTCTCTGGGGTCTTGTT 37681 TAAGAACCAGCTCAGGCTGCTGGCTCTGTTGGCAGTGGAGGTATTTCCTATAATGGGATG 37741 ATGGGATGGGTTATTCACACACATCTCAGTTACTGGGCTACATGGATCCAAATCAGCCAC 37801 CCAAGGGTTTGCAGTCACATGTGAGTCACTTAGCACAGAGAAAGAAGCCTGGAGGAGGAG 37861 GGGTCCTCCCAGCTTCAGGAGGGTTTTCCAGGATATAGGCTTCTAGTCTCGTTTTGGATC 37921 AATTTATCAGTTTTGGATTGGGTCTAATAACTCTTTCCTGAGCCTGGACTGGGCTCAAAG 37981 GCATGAGTATGTGAGGGGAATTTACTAGAATTCACCTGTAGTTTCTGTATCATTCCTAGA 38041 GAAGGGGAAGTAGAGACACTGGTGATGGGAAATAAAAACAAAACAAAACCTAAATATTGG 38101 GAGCACAGAGGTCCTTGTTCCACAGCTCTTGATAGAAGTCAGGAATGTTATGTATGTACA 38161 ATTGCCCTTGAAAAGGAAAGGATGTATGACCTGTTTTTCTGTCCCGAAGGCTGGGAACTG 38221 GGGATGATTAACAGCCTGTTGATCTGCATTATCTGAAGGGCTAGGCCATATCAAGCTCCC 38281 ACAGCTAGCACTGAAGGAGAATAGGGCCTTACAAAGGGAATTCCCTCTTTGGATCGAACC 38341 TAGGAACATCTTCTGTTTTACCGCTCTCTCCTTGTTTCATCTGCAAAGGGAGGAGCTTGG 38401 TAGTGATGTTGAGGCAGGCACCACTTGTATTTTTCTAAGCCACAGAGACTGTTTCCCTAC 38461 CTTACAAACATCCCTGTGCATCACTGCAGCTCTGTCTCTTATGGCAGTGTCTCAGTTAGG 38521 GCTTCTATTGCTGCGACTAAACACCATGACCAAAAAAGCTCACACTTCCATACTCCTGTT 38581 CATTATTGAAGAATGTCAGGACTGGAGCGCAAACAGGGCAGGGTCCTGGAGGCAGGAGCT 38641 GATGCAGAGGTCATGGAGGAAGGCTGCTTACTGGCTTGCTCTCCATGGCTTGCTCAGCCT 38701 GCTTTCTTATAGAACCCAGGACCACCTGCCCAGGGATGACACCACCTACAATGGGCTGGG 38761 CGCTAATATGAGGGATCAAAGAGATGGAGTTGTGGGAGGGACAGAGGGGGAGAGCAATGA 38821 AAGAGATAATCTTGATAGAGGGAGCCGTTATGGGGTTAGGGAGAAACCTGGTGCTAGAGA 38881 AATTCCCAGGAATCCACAAGGAAGACCCCAGCTAAGACTCCTAGCAATAATGAAGAGGAT 38941 GTCTGAACGGGTCTTCCCCTTTAATCAGATTAGTGACTACCCTAATTGTCATCACAGAAC 39001 CTACATCCAGTAACTGATGGAAGCAGATGCAGTGATCCACAGCCAAGCACTGGGCTGAGC 39061 TTCGGGAGTTCAGTTGAAGAGAGAAGGGATCATGTGAGCAAGGGGGTGGGGGAAGTCAAG 39121 ATCATGATGGGGAAAACCACAGAGACAGCTGACCCGAGCTAGTGGGAGCTCATGGACTAT 39181 GAAACGCCAGACGTTGTAGACTCCCTAAGGAAGGCCTTACCCCCTCTGAAGAGTGGATGG 39241 GGGGTGGGAAGTGGGGACGCTGGGGGACAGGAGAAAGGGAGGGAGGGGGAACTGGGTTGG 39301 TTTGTAAAATGAAAAAATAGATTTTTTTTAAATAAAAAAAGAAAGTGCTTTACATCTGGA 39361 TTTCATGGAGGCATTTTCTTAACTGAAGCTCCTTCCTCTCTGGCGACTCTAGTTTGTGTC 39421 AAGTTAACACAGAACCAGCCAGTACAGGCAGCAGAAATACCTTGCAGAAATATCTTAGTT 39481 CAGGAGTCCACGGTGGTCTCAGTCACTTCCTCATGTGCCACCTGAGTTTAACATTCCCCA 39541 AAACTTGGAACACAGGCCACCACATCATGGAGCCCTGGCTTAAAGCTCAAGTTTTATGGT 39601 ATTTTCTTTTATCACTGTCTATAATTCCTAAACATGCTACAATGTTGTGAGCCCTCACCG 39661 TCTCCTAGGTCCATAGTGACTTCCTGGCATTAATAGACTGTGCCCCAAGAGCTCTATGGC 39721 CACGACCACCACCTGCCATTCCCCTCCCCCTCCATGGTCCCAGCCTCACTTCTTCACTTC 39781 CTGGTCCTTCCGAGCCCAATGTGCAAACCCACAGAATCTGTCTGCTTATGTAAGTTTCCT 39841 GGTCACTGAGTGGGGTGACTCAGCACCAAGGTGGTGCCCTGCGATTTCCCAGCCCCAGGC 39901 AGCAGAACAACTGAAATGGAAAACAAGTCCCGTTAATAGGGTCCAGCTGAGAGCCTCCCT 39961 TTCTCAGGGAGTCTGGCAAATCTACTCCTCGGGGAACTGCCCTGGGCAGTGGAATTCTCC 40021 AGCTCCCTGCTCATTTCCTAGTTCCTCTTCCCTCTTCTCACCTTTGGCTGAGGATCAGAA 40081 AGGTTCCCACTGAGGTCTGCTTTGCCCTGGGCCTGCTCTTTTCAGAGTCCCATTTTTGGA 40141 ATGAATTTTTTTTGTCTCCTACTTTCAAGTTCACATATTGAAGCCATTATTGCCAAGGTG 40201 ATGGTATCAGAAGGAGGGACCTTTGGGAGATGAATGGATGGATTCCAAGAGGTTATGTGG 40261 GCAGAGCACCCATGATGGGGTTGGTGCCTTCATAGGAAGAAGACACAGTAGAAGGGAAAG 40321 AGATGCCGACTGAAAAACAGGAAGTCTCCTGGAGTAGGCCACTCAGCCTATGACACGCCA 40381 GCACTCAGATCTCGGACTTCCCATCTCCCAAATGGTGATAAACAAATGCTGTTGTCCAGG 40441 CTGCACAGTCTACGGCATTTTGTTGCAAGGGCCTGGACCAACCAGGCTCAGGCAGGAAGT 40501 GAATCTAGTGTGGGAGGATGTACAGACTGCCACTCAGTCTGGACACAAACTGTCCTCAGG 40561 GATCACCTGAGCCACATCTACCTAAGAATGGCTATTCTTTCCATTTGTTAACATCAAATG 40621 CCAAGCCCCTACTGTATGTAGGCTCTTGCTAGCAGTGGATATGATGCTATGTGAGATGGG 40681 AGCAATCCTCTCTGCACAGAACTATACATAGAACTATGCATAGAAGACCAACAGGGAGAC 40741 ATCAGATAACTATTAACTGTGATAGCTCTGTGGGAGACAAACAGAATGAGGGAATGGACA 40801 ATGACTTTGAGGAAAAACTATGATTGAAAATACTCTATCTGGCTGGGCGGTGGTGGCGCA 40861 TGCCTTTAATCCCAGCACTTGGGAGGCAGAGGCAGGTAGATCTCTGTGAGTTCGAGACCA 40921 GCCTGGTCTATAAGAGCTAGTTCCAGGACAGCCTCCAAAGCCACAGAGAAACCCTGTCTC 40981 AAAAAAAACAAAACAAACACACAAAAAAGAAAATATTCTGTGAGGTAAACAAGCATCTGG 41041 AAGGGTTGGGAGATAATGCAGGCAAAAATGCATTAGACAGCACACAGTACAACACAGCAA 41101 TCAAACTTAATATAAACACAGCAAATGTCATCTTTGGGCTTTGCCCCATTTCCTGATCTG 41161 ACCATAACAGCCTAGTGTCTGGAAAGCACACTAAAGCCATTTACGTCACACAGGAGTTCA 41221 ATGTTGAGTTCAGAGGGAGGGGGTGGAGGGCAGATTAGCGAGGTACAAGTTCTGGTCCCT 41281 TTGATGAAGTGTTGATGTACCCATCGACACCACACAAATATACCATCATGCTCCATGTTA 41341 GGGTCAGTGAAGGATTGCATATGTGACGGTGGCCCACTGGGCTGAGAAAGCCCTATTGCT 41401 TAGTGACATCTGTGATAATGACATGCGAGCCCTATTGCTTAGTGACATCACTCTTCTCAT 41461 AGTGTGGGATCCAATGTGTTTCTTGTACACTTGTGATAATGACATGCAAACAAGTCTATT 41521 GTGCGGCCAGTCACACAAAAAATATATTATGTGCAGTCAGGAACAGTCCATAGTACTTGA 41581 TTGGGACAGCACAAGTCTGTGTTGCTGGTTCACACATTAATCATTACCACTGTTTTAGTG 41641 TGCTCCTATATATATATATTTAAAAATTACTATAAAATGATACACCGTGCTGAGCAATAG 41701 CACCTCTTATACCTTGTGTTTACTGGATGTACTCAAGCTATTTTCTCTTGTGCTTGATTT 41761 ATTTGTATTTGTATTTTTGAGAGAACCTCATCTAGTCCATGCTGGCTTCAAACTTGTTAT 41821 AAAGCTGAGGATGGCTTCGAACTCCTGATCCCCCAGCCTCTGCCTCCCAAATGATGAGAT 41881 TACAGGCATATGCTACCAAACATGACTTTTATTTATTTTTATTACTTAGGTGGTATGGGT 41941 GGTTTGAATGAGACTGTCCCCTTTGGCTTATATATTTGTAGGTGGACCTTTGGAAAGGTT 42001 TAACAGGTATGACCATAGTGGAGGCAGTGTGTCAGTAGGGGAGGTCTTTGGGGAACCCAA 42061 TACTCAATCAATTCCAAGTTAGGGCTGTCTGTCTGTCTGTCCCCTGATTGTGTCACAAGG 42121 CAGAAACTCTCAGCTACTGCTCTAGTTCTATGCCTACCCACCTGTTGCCATGGTCCCTGC 42181 CATGATGGTCATGTACTTCAACCCTTTGGATAGGTGGCCCCCAAATTAAATGGTTTCTTT 42241 TATAAGTTGCCTTGGTCATGGTGTTTTGTCATGGCGATAAGAAAGTGACTGAGACAGGTT 42301 TGTTGCTGTTGTTACAAGGTTTAGTCCAGGCATCTGGCACCACCTCTGGCCTGTGCTTGA 42361 TTCAATCATGTTACCTTTAGAAATAGCAGGCTAAAGGACATATACCTGTGTACGTATATG 42421 TGTACGTATATATTAGCTGTATAGTCTAAGTGTGCACCTGACTCTAATATCTAGGTTTGT 42481 GTAAGTAGACTCCACCAAGCTCACTAAGCAATGGTATCACAGTTTTCAGATAGTGTTCAG 42541 CGATGCTTGGCTGAGTGTTAGTTCTTTTTTTAATATTTTATTTATTTATTATGTATACAA 42601 CATTCTGCTTCCATGTATCTCTGCACACCAGAAGAGGACACCAAATCTCATAACGGATGG 42661 TTTTGAGCCACCATGTGGTTGCTGGGAATTGAACTCAGGACCTCTGGAAGAGCAGTCGGT 42721 GCTCTTAACCTCTGAGCCATCTCTCCAGCCCCTGAGTGTTTTTAAATCAAGGAAAAAAGC 42781 CTGAGGGAAGGGAGCTCAGGCTGAAGGGGAGGAGTCAAGACAGTCTGACCCCAAGGCATT 42841 GTGGGACGTAAAGAGTTCTGGGACAAGACTGAGGTCTCTTCCTTCTCAGAGACTGTGGGC 42901 TTCAGTTTCCTTGGTAGCCGGAAGCAAAGCTAATCCATGGCTTAAAATATAATACTCAGT 42961 GTAACCTTGTGTTGTAGAAGTGACTTGCTTGTCTTCTTCCATAATTCTAAAACATCTTTA 43021 AGAGCAGGATCCAGGAAGGGAAAAGGAGAGATTCTCATCTTCTTCAAAAGGCAGCTTTCC 43081 CTAAAGCATTTTCTGATGAAATTTAAGTTCTAAAACCAGCAGTGGTATAATCCCATCATG 43141 AATGGGGATCTCTGAGTTTAAGGCCAGCCTGGTCTACAGAGCAAGTTCCAGGACAGCCAC 43201 GGTTACACAAAGAAATCCTGTCTTAAAACAAAACAAAACCCAAAACAAACATAAACAAAA 43261 ACTATCCAAAACCAACCAACCCCCCCAACTCAGAAAGAAAGAAAGAAAGAAATCAAGAAA 43321 GAACTGCCCACCGGGTGTTGGTGGTGCAAGCCTTTAATCCCAGCACTCGGGAGGCAGAGG 43381 CAGGCAGATCTCTGTGAGTTTGAGGCCAACCTGTTCTCCAGAAAGAGTGCCAGGATAGGC 43441 TCCAAAGCTACACAGAGAAACCCTGTCTTGAAAAAAGAAAAGAAAGAACTACCCATGACC 43501 AAACAGTTCCATGGCCAGGTAGAGAATGAGGACGCTGAAAGTCACACCTTCTCAGAGTCT 43561 CAAACTGCACATCTGGCCTCAAAGTCCAGAAATGAGTGCAAGACCATTAATGACAGTCTT 43621 TGGAAACAAACCAGACCAAAGAACATTTGGCTCCTGATACATATTCTGAGGGTCACATAG 43681 AAAGAAAGATCTGCCTTTGGCCACCTCCTTTTGAAGTGGGGAATTTTATTTTCTTCTGCA 43741 TGGAAACTTCATGTAGGTATTTGAGAATACATACAGACATGCAGGTGCACATGCACGGAC 43801 ATGAACACACACATACACCCCGGGTAGGCAGGCAAGAAAGTGTGTGGAATAACACTTGAA 43861 CTTCCCTTCCAGAACAGAAGCCCTCTGAAGTGTGACATTCATGCTGGCTGCATGGGGTCT 43921 GATCAGTACTAGTGAGTGGAGGTGGAGGGGTAGGAAACATGGGGATGATAATAGGTTGTC 43981 AGGAAAGTGGTGCCCCAGGTAGCACAGAGTAGAAATTTGTCCCCCAAAATCCTTTTGAAC 44041 CCAGTTGATTTGAATGCCGTGCCCCTGCCACCCAGGCTTCAGAGCTAAGTGACTTATGTC 44101 TTCAGGTCAGTGATGATTACCACGGTTGCAGTGCTAACACAGATGCTTTATCTACCAGGA 44161 CAGAAACAAGAAAGATGCTCCTTCCCAGGCCCCTTAGCACTCTCTGGGTGGGGAGGATTG 44221 CCCCACCTTCCAAAAATAGAATACTGTTTTGGTAAACAGCCACTTTGAGCCCATGAGGAT 44281 ATCTTCATTAGCTATGGAGACAGGTTTTAGTAAGAAAGCAAGATGAGAGGCTAAAAAACC 44341 CTTGGGGAGCAGGAACTGGGAAGACTGTGGTACCTTGTTCCCAGATCCACCAGAAACCTT 44401 GCCACCAGACGATGTGTCCAGGCCCCACATATTTCACAAAAAGTTGGATCTGATAACAAT 44461 GAGGATGGAATCCCGGTCTTAAGGTGGGTTTGGGGTGGGAAGAGGCGGGATAATGGGTGA 44521 GAGGGTCGGTGGGGACAGGTGAGATGGGGTATGGTGGGGAGAGGTGGAATGGGGTGGGGT 44581 GGGTTGAGATGGAGTATGGTACAGCGGGGAGGGATAGAATTGTCTTTTCCCTGTACCACA 44641 GAGAAGTTTGACTGCTACCCTTGGCAATTAATCAATTATAGAAAATGCAACTTTGCTTTT 44701 AAAATGTGTCTATTTCCAAAGGCTTCTTCCCCTCCCCTACCTAGGGAGAAGGAAAGAATG 44761 GATAATGCTACTGTAGAGGAGGGTAGCATCACTATAGAGGCCTCAGTATCTGCCCCAGGG 44821 AGCTGGGAGAGAGTTCTATCACACAAACACAGCCCGAGTCACATACTCAACAAACCCCAC 44881 AAAACAAAACAACAATAATGAAGATACAAAATCTCATTATGTAGCCCAGGCTAGTCCTAG 44941 ATTTCTGTTTTCTTTTTTTGTTTTTCGAGACAGGGTTTCTCTGTGTAGCTTTGGAGCCTA 45001 TCCTGGCACTTGCTCTGAAGCCCAGGCTGCCCTCACTCACAGAGATCCGCCTGCCTCTGT 45061 CTCCAGAGTGCTGGGATTAAAGGCGTGCACCACTAATGCCTGGCTAGTCCTAGATTTTTT 45121 TATCCTCCTGCCTCAGGCTCCCAACTGTTGGGTTTACTTTTGGGAGTCCATTTTCTTCCA 45181 GCATGGATTCTTTGAATTGAAATTCAGATTATCAGGTTTCTGTAGCAATCCCACCAGCCC 45241 ATTTTTTTGTCTGACACTGCTTGTTTTGAGACACAGTCTCCCACTGCTGTAGCCCAGGCT 45301 GCCCTAGATTTTCTATGTAGCCCAGGCTGGCCTTGAACTCCCAGGAGTCCTCTGGCCTCT 45361 CCCTTTTGATTACTGGAACTAGAAGAAGTCACTATGCTTGACTTGGAACTAATATTAGAA 45421 CAAAATATATTTTTCATTGAGATTCAACTTTGAAATCCTGATGCTCCTGCCTCACTCAGG 45481 TCATCAGGGTTGGCAGCAAGAGCCTTTATCCACTGAGTCATATTGGGCCCTGACCTGCTT 45541 TTAAATTTTGCCTTTAGGGCTGGAGATGTAGCTCGGCTGGTTCAGTGCTTGCCTGGTACC 45601 CACGAAGCCCTGGGTTTGATCTACAACACAGTATAAGCCAGGCCTGATGGCGTATACATG 45661 TAATCCTAACACTTGGGGAGCAAGAGGGAGGCCAAAGCCATCCTCTGCTACTTGGTGAGC 45721 TTGAGGCCAGCCTGGGATCCTTGAGACCCTGTTTCAAAACAATAACAACAAACACAGACT 45781 ACTAAAAAAAATTAATAAGGGCCAGACTGGGTGGTGTATTCCTTTAATCCAAGCAATGAG 45841 GAGGCAGAGGCAGGCAAATGTCTGTGAGTCTGGGGACAGCCTGGTCTACTGAGCAGCAGG 45901 CCAACTAAGGCTACATAGTGAGACTATCTCAAAAAAAGCAAAATAACAATAAACAGACCA 45961 GTTCCCCATCTCCTATTTTGCCTTTACCTCCTATTCCCTGCTCAGCAGGTTATTTTTTGT 46021 TCCTGCATCTTGGTTCACTGATCTGTAAACTTGTCTGAATAAGTAGGTACAGGGTTGTTT 46081 TAAAATTAGATAATATATTCAATGAGAAGGGCTACCAAGTGCTCAACCAATGTATGCATA 46141 TGTATGTATGTATGTATGTATGTATGTATTTATTTTTGTTTTGTTTTTCAAGATAAGGTT 46201 TCTCTGTGTAGTTTTAGAGTCTGTCCTAGAACTTGCTCTGAAGAGCAGGCTGGTCTTGAA 46261 CTCACAAAGATCCACCTGTCTCTGCCTCCCAAGTGCTGGGATTAAAGGCATGTGACACCA 46321 CCCCCAAAGCCAATGTTCTTATAGGCATCTTTGATTTTTTTTCTCTTTCTTTGAGTGGAG 46381 TCTGACTAAGTAGCCCATACTAGCTCTGCATTTACAATCTGAACACATGGATAAGAGTGG 46441 TGAAAATTATCAAGATCATGTTATGCTATGCCTCCTGAGTCACCATGCCCTGCTTCAGAC 46501 TTCTTTGTATTAAAGAACTGTGTAAAAAAAAAAAAAAAGACATTTGAAGGCACATAATCA 46561 GAGGAATTTGTCAGTGATTTTTCACATACTGTCTTATTTGTGGCCAAGGTAAGCCTAGAG 46621 AGTATTTCTTAAAATTAAAAATAGTGGGCAGATTTTGGAGGCGATCTGATATGAAAATCC 46681 CTTCCCACCCCAGGTAGTCATGGGCTGACTATCAAGGATACATTCTGAGACATATATCCT 46741 CAAGCAGTTTCTGCCTTACGCAAATATCATAGGTCATAGCACACTGAGACTATGTGGCAG 46801 TCTATGTGTCTATATACACATGGTGTGGCCTATTGTTCCCATGGTCACAAAGAACAAAAC 46861 AACTTTTTCACAAGGCTTTACCCCTAGAGGAAGAGCTACAGGCAATCAATGGTTGCTGAG 46921 AGGAGTATCAGTCTTCTCCAGGGACTTAGCCAATCCCAAGAGGTCAGCCACGCATAGGAA 46981 CGCTTAGCCACGCTTGTATAGAACATCTCAAACAACAACCACCTCAGTGTAAAGCAAGCA 47041 CACAAGGAACTGATGCAACTAAGAGACAAAGGGCCCGGTGTGTGTGGCCCGTAGCTGTCA 47101 TCCCAGCACTTGAGACTAAGGAAGGAAGGTTGAGAATTTGAGGCCAGCATGGACTCCACA 47161 GAAAGACCGTTTTCTTTCTCAGAAAAAAGAAGCAAAAACCAAGAACAAGGTGTATGGGAA 47221 TGCTACTGTCTTGGCATATTGTTTATAGAAAACTTTTTTATATATAAAAGGAATGCACTA 47281 CAAAAATTATAAACTACTGTAATATTAACTGCATAGATCTATAACATGGTCATTTATTAT 47341 TGAGTATGATTATCTATCTACCCACGCTGCAGGTTTAGACAGTTGCACTACAGTAGATCT 47401 GTTTGCAGTAGCATCATTATTAGACATTTTGGACAAAGCCAAGTGGTAATGGCACATGCC 47461 TTTAATCCCAGCACTTGGGAAGCAGAGGTAGGCGGATCTCTGTGAGTCAGAGACCAGCCT 47521 GGTCTACAAAGAACTAGTTCCAGGAGAGTCTCCAAGGCCACAGAGAAACCCTGTCTCGAA 47581 AAACCAAAAGAAAAAAAGAAAACAAAAAACTAAAAAATAAATAAATTTGGGGCAATATCT 47641 TGTCCTATGATGTTACTGGGTAATGGGATTTCCTCCTCTTGTATTATTTTTTCTTTGGGG 47701 GTTTTACTTATTATTTACTTGAGACAGAGTCTCATTTATGACAGGCTGGCCTCAAACAGG 47761 AAATGAAGCCAAGGAAGACCTTGAAGACCTAATCCTTCTGTTTCTTCCTCCTATATGGTG 47821 AGTTAAAGGCATACAGTACCATGCCCAGTCTATTCACTGCCCAGGGCTTCATGCATGCTA 47881 GCAAAGCACCAACTGAGCTGCATCCCCACCCCTCCTCCTGGCTTCCATCTCCTTATGTAG 47941 CTAGAAATGAGCCTGTCTGTCTCAAATACTGGGATTATGGGTGTGTGCCACCACACCTGG 48001 CTTCCTATTATAGCCTTGTGGGATCACTGTTGTTTACTGAAGCATTGTGACACACTGCAG 48061 ATTGCTGGAACAGCGTCTGCCATCATCATGACACAACTTCAGAGAAAGAGAGAGTTCCCA 48121 ACCAGCCACACACTTAACTCAATGCCTGTAGCCCTTATTCTGTTAAGACGATTTCCTGCC 48181 ATCTTACTCAAAGACCCTCTTTAACTCGGTAGGAACATCTGTTACACTGAAAGTCCTGCC 48241 TGTTGCTCCACTGACCTCCTTCACAAATTATTATATTTTGGAGCCAATTCTGAACCCAGG 48301 TTTTCTGAGTGACACATTTTAGTATTTTTTTTTTCTTTCTATTTTCTTTCATGGAAAGTC 48361 TCTTGTTACTGTTCACATGACCAAGGATCACTGCATCATCTTCCAAGGCCAATTTTGGAT 48421 GTTTCAGCAAGGGAGACTGAAGATCCTGAGTCTCAGTGTTGATCTCCTTTAGAATGTCCT 48481 CTGGAGAAGGTAGTGACAACACTGCAAGGATAATAGGTGAATAAAGGGAAGCCAGAGTGT 48541 CCTCTGGGATGTGCGGCACTTACATGAAGGATTCATTTATAAATTTTAAGTTATGGAGTA 48601 TAATAATAAGACTAAATATGTAGTGTCGTAATTTTATAACTATACATATGTATATAGTAA 48661 ATATAAATTTATATGTAATGTATTTATAGTAAGTGTACATAGAATTGAACATATGTTACA 48721 TAAATGGCAGAAAGGAATGATTCTCAATTGCTTTTTTTCTAATTATAATTTCTATTGCTC 48781 TTTGTGGATTTCACACCATGCATTCTGATCCCACTTATCTCCTTGTCTCCTTGCATTTGC 48841 CCTCTGCCCTTGCAACCTCACCCCCAAATCAAAGCCAAATTTAAAAAAAAAACCAAAATC 48901 CAAACAAAACAGAGACAAAACAAAAATAAAAGCAACAACAAAAAAAGGAGAATCTTGTCA 48961 TGGTAGCTGTAGTGTGGCCTGTTGAATCACACAGTATACCCTTTAGTCCATTCATCTTTT 49021 CTTCCAAGTGTTCATTGATACAAGTCACGGTCTGGCTCGAGGATTCTGGTTTCTGCTATA 49081 TTACTAATAATGGGCTCTCACTGGGGCTCCCCTTGGATATCCTATTGTCCTGTGTTATGG 49141 AGAGCCTGCTGTTTTGGATATGTAGGTTTGTCCCCTTCACATGCTATAACAATTCATAAA 49201 TTCAGTGAATGTTGGGGTGGGCCAACTCATAGCCCTGGTTCTGGGCTTGGGTGGTATTAT 49261 TAAACCCACTGATGGAGAATAAGACCACTACCATAATTTAAAAGCCAAATTGAAGCAAGT 49321 TTTAATTCAATACTGCCCAGGTGGACAGGCTCTGGCTAGGTCCATCTCTGAGTTTCCAGG 49381 AGGTGGCCCTGACTCACGGTTTACAGTGGCTTGAGTATTTTCCATAAGGTCCAATCAGGG 49441 GCAAGCATACATCCTGATGTACCTCCAGTCTATATCCAATCGGGGGCAAGTGTACATCTT 49501 GATGTATTTCCTGCCTGTGAACCTACTGCCCACATGTGATCAAGCACATCCGGTGCAGTT 49561 GGGTCAAACAGACTTGTTTAGGGCAATGAAAAACACATGGCTTTTTATCTCCCATAAACA 49621 ATAGCCTCCAGCGGTTCAGGGACTATTTGTCCTTGGGCAAGGAATTTACAGATCCTATAG 49681 GTGAGTCAGGGTCAGCATCCTGCTCTCATGCCCTCAGGGCTGGCTCACTTGTTACCTCCC 49741 CGACCCTCTCTCAACAGGGTCAGCTCTGAGGTGCTGCCCAGGTGGGGTGCAGGGCCTACT 49801 CTTCCGCATGTTGCAGCTGGTCAGGGTTAGTTCTCTCATATGCCACAGGTGGCAATGGGT 49861 GAAGGGGGAGGGCATGTTTCCCTCATCAACGCCATTACATGGGGGGATGGGGTCAGCTCT 49921 CATGCCCTTAGGGTTGGCTCACCTGCATCCTTGACCATAGGGTCAGCTCTAGTATGCTGC 49981 TCAAGTGAGGCGCACACCTA SEQIDNO:4(LoxPsequencefrombacteriaphageP1) 1 ATAACTTCGTATAGCATACATTATACGAAGTTAT SEQIDNO:5(FRTsequencefromthe2mplasmidofthebakersyeast Saccharomycescerevisiae) 1 GAAGTTCCTATTCtctagaaaGTATAGGAACTTC SEQIDNO:6(attBsequencefromE.coli) 1 cCTGCTTttTtatActAACTTGa SEQIDNO:7(RecognitionsitefortheCHO-23/24meganuclease,35,699 basepairsdownstreamofCHODHFR) 1 TAAGGCCTCATATGAAAATATA SEQIDNO:8(RecognitionsitefortheCHO-51/52meganuclease,15,898 basepairsdownstreamofCHODHFR) 1 ATAGATGTCTTGCATACTCTAG SEQIDNO:9(CHO-23/24meganuclease) 1 MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSIKAQIFPNQCYKFKHQLRLRFQVTQKTQ 61 RRWFLDKLVDEIGVGYVTDRGSVSDYMLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIE 121 QLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSA 181 ASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSIIAQIKPGQSYKFKHTL 241 QLVFQVTQKTQRRWFLDKLVDEIGVGYVIDRGSASDYRLSEIKPLHNFLTQLQPFLKLKQ 301 KQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKK 361 KSSP SEQIDNO:10(CHO-51/52meganuclease) 1 MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSIIAQIPPNQSCKFKHQLRLTFQVTQKTQ 61 RRWFLDKLVDEIGVGYVRDRGSVSDYILSEIKPLHNFLTQLQPFLKLKQKQANLVLKIIE 121 QLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSA 181 ASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSIYAGIAPNQSCKFKHQL 241 RLWFVVSQKTQRRWFLDKLVDEIGVGYVIDNGSVSHYRLSEIKPLHNFLTQLQPFLKLKQ 301 KQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKK 361 KSSP SEQIDNO:11(CHO-51/52donorplasmidwithEcoRIsite) 1 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA 61 CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG 121 TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC 181 ACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCC 241 ATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTAT 301 TACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGT 361 TTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCAGAAAC 421 CTTTCAACCAGCTTTTGAGCTAATGATAGAGAGAAGCTCAAGGAATTGGAGCAATGCTTG 481 ACTAGGGATGTCAGAGGGAGGCTATCCAGAGGAGCTTACAACTGAGGTAAACTTAAAAGT 541 TAGGGAGTTTGTCAACTTCAACCCACAGAATAGAGCAGAGCCAGGAGGAGCTGAGGCTTC 601 TGAGTGTTATGGTGGAAGCATCACCCCAACCCTTGACATCCATATGCCTGAAGAGTCTGG 661 AATGTTATGGTGGAAGTTCCACCCAAGCCTCCCTTCCCGGTCGCCCTCCAAACCCTGCTA 721 CATCTCAGAAATCCCACCAAATGATGACTCCCTCCCCCAGAGATATTCAAGACCACTCCC 781 ACAGGGTATTTAAACTGCCCCCCAACCCCCAGAAAATAGATGTGTGGTTTTCCAATCTCT 841 CTTTCCTATCACGTCTCTGGGGAGCTGGCAGGCCATTTGGGAGCATTGTATCCATTAAAC 901 GACTTCTCAGTGGAGACTCTGAAAGCCAGAAGAGCCTAGACAGATAGATGTCTTGCGAAT 961 TCTTGCATACTCTAGAGACTACAGATGCCGGCCCAGACTATTATATCCAGCAAAAGTTTC 1021 AAACACCATACAAAGTCAAATTTAAACAGTATCTATCTACAAATCCAATATTACAGAAGG 1081 TGCTAGTAGGAAAACTCCAAACTAAGATTAACTATACCTGTGAAGACACAGGAAATAATC 1141 TCACACTGGCAAAAGAAGAAAAACCTCTCTCTCTCTCTCCTCTCTCTCTCTCTCTCTCTC 1201 TCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCACACACACACACACACACACACACACAC 1261 ACCAACACCAATACCATGAACAACAAAATAACAGGAATTAACAATAATTGATGTGTGTGT 1321 ATGTCCCTGTGTGTGTGTCCTTGTGTGTGTCTGTTTGTGTGTCTGTGTATATGTTTGTCA 1381 CCTGAGGGGTGGCTCTTCCTTGGTTTGTGAGGTTTCTACCCAAAAGCTTGGCGTAATCAT 1441 GGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAG 1501 CCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTG 1561 CGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAA 1621 TCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCA 1681 CTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGG 1741 TAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCC 1801 AGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC 1861 CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGAC 1921 TATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCC 1981 TGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATA 2041 GCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGC 2101 ACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA 2161 ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAG 2221 CGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTA 2281 GAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTG 2341 GTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGC 2401 AGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGT 2461 CTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA 2521 GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATAT 2581 ATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGA 2641 TCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATAC 2701 GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGG 2761 CTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTG 2821 CAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTT 2881 CGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCT 2941 CGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGAT 3001 CCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTA 3061 AGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA 3121 TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAAT 3181 AGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCAC 3241 ATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAA 3301 GGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTT 3361 CAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCG 3421 CAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAAT 3481 ATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTT 3541 AGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCT 3601 AAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTC 3661 GTC SEQIDNO:12(RecognitionsitefortheCHO-13/14meganuclease,inIntron2of CHODHFR) 1 TACATGTATGTACAAAATATAT SEQIDNO:13(CHO-13/14meganuclease) 1 MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSIFASITPRQCYKFKHELQLTFVVTQKTQ 61 RRWFLDKLVDEIGVGYVIDQGSVSHYRLSEIKPLHNFLTQLQPFLKLKQKQANLVLKIIE 121 QLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSA 181 ASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSIIAQIKPNQSCKFKHQL 241 MLTFTVAQKTQRRWFLDKLVDEIGVGYVIDIGSVSEYRLSQIKPLHNFLTQLQPFLKLKQ 301 KQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKK 361 KSSP SEQIDNO:14(RecognitionsitefortheCGS-5/6meganuclease,inExon4of CHOGS) 1 AAGGCACTCGTGTAAACGGATA SEQIDNO:15(CGS-5/6meganuclease) 1 MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSIKAIIRPEQSYKFKHRLRLVFQVTQKTQ 61 RRWFLDKLVDEIGVGYVYDRGSVSDYYLSEIKPLHNFLTQLQPFLKLKQKQANLVLKIIE 121 QLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSA 181 ASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSIWARIKPGQSYKFKHTL 241 ELVFQVTQKTQRRWILDKLVDEIGVGYVTDAGSASVYRLSEIKPLHNFLTQLQPFLKLKQ 301 KQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKK 361 KSSP SEQIDNO:16(ForwardPCRprimerforevaluatingCHO-23/24targetsite) 1 ggagggacattaatctgcatgcagtgatc SEQIDNO:17(ReversePCRprimerforevaluatingCHO-23/24targetsite) 1 gtcttggtttgggttgtctaagcaacctc SEQIDNO:18(ForwardPCRprimerforevaluatingCHO-51/52targetsite) 1 CACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGG SEQIDNO:19(ReversePCRprimerforevaluatingCHO-51/52targetsite) 1 CGATGGCCCACTACGTGAACCATCACC SEQIDNO:20(PCRtemplateformRNAencodingCHO-23/24) 1 CACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTC 61 ACTATAGGCTAGCCTCGAGCCGCCACCATGGCACCGAAGAAGAAGCGCAAGGTGCATATG 121 GCACCGAAGAAGAAGCGCAAGGTGCATATGAACACCAAGTACAACAAGGAGTTCCTGCTC 181 TACCTGGCGGGCTTCGTCGACGGGGACGGCTCCATCAAGGCCCAGATCTTTCCGAACCAG 241 TGCTACAAGTTCAAGCATCAGCTGAGGCTCCGTTTCCAGGTCACCCAGAAGACACAGCGC 301 CGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGGGTGGGCTACGTGACTGACCGCGGC 361 AGCGTCTCCGACTACATGCTGAGCCAGATCAAGCCTCTGCACAACTTCCTGACCCAGCTC 421 CAGCCCTTCCTGAAGCTCAAGCAGAAGCAGGCCAACCTCGTGCTGAAGATCATCGAGCAG 481 CTGCCCTCCGCCAAGGAATCCCCGGACAAGTTCCTGGAGGTGTGCACGTGGGTGGACCAG 541 ATCGCGGCCCTCAACGACAGCAAGACCCGCAAGACGACCTCGGAGACGGTGCGGGCGGTC 601 CTGGACTCCCTCCCAGGATCCGTGGGAGGTCTATCGCCATCTCAGGCATCCAGCGCCGCA 661 TCCTCGGCTTCCTCAAGCCCGGGTTCAGGGATCTCCGAAGCACTCAGAGCTGGAGCAGGT 721 TCCGGCACTGGATACAACAAGGAATTCCTGCTCTACCTGGCGGGCTTCGTGGACGGGGAC 781 GGCTCCATCATCGCCCAGATCAAGCCGGGTCAGTCCTACAAGTTCAAGCATACCCTGCAG 841 CTCGTTTTCCAGGTCACGCAGAAGACACAGCGCCGTTGGATCCTCGACAAGCTGGTGGAC 901 GAGATCGGGGTGGGCTATGTGATCGACCGCGGCAGCGCCTCCGACTACCGCCTGAGCGAG 961 ATCAAGCCTCTGCACAACTTCCTGACCCAGCTCCAGCCCTTCCTGAAGCTCAAGCAGAAG 1021 CAGGCCAACCTCGTGCTGAAGATCATCGAGCAGCTGCCCTCCGCCAAGGAATCCCCGGAC 1081 AAGTTCCTGGAGGTGTGCACCTGGGTGGACCAGATCGCCGCTCTGAACGACTCCAAGACC 1141 CGCAAGACCACTTCCGAGACCGTCCGCGCCGTTCTAGACAGTCTCTCCGAGAAGAAGAAG 1201 TCGTCCCCCTAGACAGTCTCTCCGAGAAGAAGAAGTCGTCCCCCTAGCGGCCGCTTCGAG 1261 CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAA 1321 AATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCA 1381 ATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGT 1441 GGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGATCTTGA 1501 TCCGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAG 1561 CCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTT 1621 ACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTC 1681 CCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT 1741 TTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGAT 1801 GGTTCACGTAGTGGGCCATCG SEQIDNO:21(PCRtemplateformRNAencodingCHO-51/52) 1 CACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTC 61 ACTATAGGCTAGCCTCGAGCCGCCACCATGGCACCGAAGAAGAAGCGCAAGGTGCATatg 121 gCACCGAAGAAGAAGCGCAAGGTGCATATGAACACCAAGTACAACAAGGAGTTCCTGCTC 181 TACCTGGCGGGCTTCGTGGACGGGGACGGCTCCATCATCGCCCAGATCCCGCCGAACCAG 241 TCCTGCAAGTTCAAGCATCAGCTGCGCCTCACCTTCCAGGTCACGCAGAAGACACAGCGC 301 CGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGGGTGGGCTACGTGCGCGACCGCGGC 361 AGCGTCTCCGACTACATCCTGAGCGAGATCAAGCCTCTGCACAACTTCCTGACCCAGCTC 421 CAGCCCTTCCTGAAGCTCAAGCAGAAGCAGGCCAACCTCGTGCTGAAGATCATCGAGCAG 481 CTGCCCTCCGCCAAGGAATCCCCGGACAAGTTCCTGGAGGTGTGCACCTGGGTGGACCAG 541 ATCGCCGCTCTGAACGACTCCAAGACCCGCAAGACCACTTCCGAGACTGTCCGCGCCGTT 601 CTAGACAGTCTCCCAGGATCCGTGGGAGGTCTATCGCCATCTCAGGCATCCAGCGCCGCA 661 TCCTCGGCTTCCTCAAGCCCGGGTTCAGGGATCTCCGAAGCACTCAGAGCTGGAGCAGGT 721 TCCGGCACTGGATACAACAAGGAATTCCTGCTCTACCTGGCGGGCTTCGTGGACGGGGAC 781 GGCTCCATCTACGCCGGGATCGCGCCGAACCAGTCCTGCAAGTTCAAGCATCAGCTGCGC 841 CTCTGGTTCGTGGTCAGCCAGAAGACACAGCGCCGTTGGTTCCTCGACAAGCTGGTGGAC 901 GAGATCGGGGTGGGCTACGTGATTGACAATGGCAGCGTCTCCCATTACCGCCTGAGCGAG 961 ATCAAGCCTCTGCACAACTTCCTGACCCAGCTCCAGCCCTTCCTGAAGCTCAAGCAGAAG 1021 CAGGCCAACCTCGTGCTGAAGATCATCGAGCAGCTGCCCTCCGCCAAGGAATCCCCGGAC 1081 AAGTTCCTGGAGGTGTGCACCTGGGTGGACCAGATCGCCGCTTTGAACGACTCCAAGACC 1141 CGCAAGACCACTTCCGAGACTGTCCGCGCCGTTCTAGACAGTCTCTCCGAGAAGAAGAAG 1201 TCGTCCCCCTAGACAGTCTCTCCGAGAAGAAGAAGTCGTCCCCCTAGCGGCCGCTTCGAG 1261 CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAA 1321 AATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCA 1381 ATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGT 1441 GGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGATCTTGA 1501 TCCGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAG 1561 CCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTT 1621 ACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTC 1681 CCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT 1741 TTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGAT 1801 GGTTCACGTAGTGGGCCATCG SEQIDNO:22(PCRtemplateformRNAencodingCGS-5/6) 1 CACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTC 61 ACTATAGGCTAGCCTCGAGCCGCCACCATGGCACCGAAGAAGAAGCGCAAGGTGCATATG 121 GCACCGAAGAAGAAGCGCAAGGTGCATATGAACACCAAGTACAACAAGGAGTTCCTGCTC 181 TACCTGGCGGGCTTCGTCGACGGGGACGGCTCCATCAAGGCCATTATCCGGCCAGAGCAG 241 TCCTACAAGTTCAAGCATCGCCTGCGGCTCGTTTTCCAGGTCACGCAGAAGACACAGCGC 301 CGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGGGTGGGCTACGTGTACGACCGCGGC 361 AGCGTCTCCGACTACTATCTGAGCGAGATCAAGCCTCTGCACAACTTCCTGACCCAGCTC 421 CAGCCCTTCCTGAAGCTCAAGCAGAAGCAGGCCAACCTCGTGCTGAAGATCATCGAGCAG 481 CTGCCCTCCGCCAAGGAATCCCCGGACAAGTTCCTGGAGGTGTGCACGTGGGTGGACCAG 541 ATCGCGGCCCTCAACGACAGCAAGACCCGCAAGACGACCTCGGAGACGGTGCGAGCGGTC 601 CTGGACTCCCTCCCAGGATCCGTGGGAGGTCTATCGCCATCTCAGGCATCCAGCGCCGCA 661 TCCTCGGCTTCCTCAAGCCCGGGTTCAGGGATCTCCGAAGCACTCAGAGCTGGAGCAGGT 721 TCCGGCACTGGATACAACAAGGAATTCCTGCTCTACCTGGCGGGCTTCGTGGACGGGGAC 781 GGCTCCATCTGGGCCCGGATCAAGCCGGGGCAGTCCTACAAGTTCAAGCATACCCTGGAG 841 CTCGTGTTCCAGGTCACCCAGAAGACACAGCGCCGTTGGATCCTCGACAAGCTGGTGGAC 901 GAGATCGGGGTGGGCTACGTGACCGACGCCGGCAGCGCCTCCGTCTACCGCCTGAGCGAG 961 ATCAAGCCTCTGCACAACTTCCTGACCCAGCTCCAGCCCTTCCTGAAGCTCAAGCAGAAG 1021 CAGGCCAACCTCGTGCTGAAGATCATCGAGCAGCTGCCCTCCGCCAAGGAATCCCCGGAC 1081 AAGTTCCTGGAGGTGTGCACCTGGGTGGACCAGATCGCCGCTCTGAACGACTCCAAGACC 1141 CGCAAGACCACTTCCGAGACCGTCCGCGCCGTTCTAGACAGTCTCTCCGAGAAGAAGAAG 1201 TCGTCCCCCTAGACAGTCTCTCCGAGAAGAAGAAGTCGTCCCCCTAGCGGCCGCTTCGAG 1261 CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAA 1321 AATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCA 1381 ATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGT 1441 GGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGATCTTGA 1501 TCCGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAG 1561 CCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTT 1621 ACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTC 1681 CCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT 1741 TTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGAT 1801 GGTTCACGTAGTGGGCCATCG SEQIDNO:23(ForwardPCRprimerforevaluatingCGS-5/6targetsite) 1 tgacagctctggccttaagtgcctacgaaactag SEQIDNO:24(ReversePCRprimerforevaluatingCGS-5/6targetsite) 1 gtctttcctctttgctgtagccttggtagaactactgcc SEQIDNO:25(CHO-23/24Insertiontargetsequencedonorplasmid) 1 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA 61 CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG 121 TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC 181 ACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCC 241 ATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTAT 301 TACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGT 361 TTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCCATACCCAGGGGAGCTGTA 421 CTGGGCTGCAGCCCTGCGCCATTCAGCCATGCACCAGGCTACTCCCTCCTCTTCCAGCTT 481 TCTCCTTCTGATGGCCATAGGATTAGAAGATAAGGGACTCTAGTGCAGGTCAACTGCTGA 541 CCAGTGTGAAAATGCACAGACTACATGCTGGTAGATCAGCACTTCAAACTACTGTTCACC 601 ATCATCTCTGGAATAAGCACTACATTTACAGGGTTCAAACCTCAATGAATATAAACAAAC 661 AAAACACACCTCCCTTCCTTCACTGTCTCCCATTTCTTTGGTTCCCATCTCCACATAGAA 721 TTTATAATTAAAATTTCTAAGTATCTTTCCAGAAATACTTCACACATGTTATAAGCAAAT 781 GTGCTTTTAAAGATACTATTTTAAATTATGAAAATGGTTATATTAGTTGAGATAAAAGAA 841 TAGAATGGGAAGTTCCAGAATTTAAGGCCTCATATGGATCCCAGCTGTGGAATGTGTGTC 901 AGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATC 961 TCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGC 1021 AAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGC 1081 CCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTT 1141 ATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTT 1201 TTGGAGGCTACCATGGAGAAGTTACTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGA 1261 ACTTCAAGCTTGGCACTGGGTACCGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCG 1321 CGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTC 1381 GTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTC 1441 CAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAG 1501 CTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCC 1561 ATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGC 1621 AACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACCCGAGCGAAAACGGTCTGCGC 1681 TGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAAC 1741 ATCAGCCGCTACAGTCAACAGCAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCG 1801 GAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCC 1861 TGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTG 1921 GTCTGGTGTCAAAAATAATAATAACCGGGCAGGGGGGATCTGCATGGATCTTTGTGAAGG 1981 AACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTA 2041 AGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTG 2101 TATTTTAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCCTTTAATG 2161 AGGAAAACCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACT 2221 CTCAACATTCTACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTT 2281 CAGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTG 2341 CTATTTACACCACAAAGGAAAAAGCTGCACTGCTATACAAGAAAATTATGGAAAAATATT 2401 CTGTAACCTTTATAAGTAGGCATAACAGTTATAATCATAACATACTGTTTTTTCTTACTC 2461 CACACAGGCATAGAGTGTCTGCTATTAATAACTATGCTCAAAAATTGTGTACCTTTAGCT 2521 TTTTAATTTGTAAAGGGGTTAATAAGGAATATTTGATGTATAGTGCCTTGACTAGAGATC 2581 ATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTC 2641 CCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCT 2701 TATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCA 2761 CTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCCCC 2821 AGGAAGCTCCTCTGTGTCCTCATAAACCCTAACCTCCTCTACTTGAGAGGACATTCCAAT 2881 CATAGGCTGCCCATCCACCCTACTAGTATATGAAAATATAAAGCGCTTTCTCTTTTAAGT 2941 CTAGGGTAGGTGTACTAGATCAGCGCTCAGCTCCATACCATGAAGCCATCCAGGAGTCAG 3001 ACCTCTCTGACAGCCCTGCCATTGTCACAGAGAAGTTTCTGTCACCAGTGCTCATGCTGT 3061 CAGAGGAGCGAAGGAGAAAAGATGTGAGACCTCCCAAGTCAAAGTCATCTATGGATAAAA 3121 CCTTAGTTGCATGGCACACCAGTGTTAGGGAGTCGGGGAAACACAGCCATAGCCCAGCTT 3181 CCTCTCTGTTCTTGCTCTTATTACCACCAGAAAGAGGTTGCTTAGACAACCCAAACCAAG 3241 ACACAGGGCTCTGTGGGAGGGAATCAGTCCCAGGCTTCTGGCACATGCTATGTCACCGGA 3301 AAGCCCCAGCCCTACTCCGAATCCCCACAAGTACAGCAAATATCAGATTATAGCATTTAA 3361 AGGGGCACTCTTGCCAAAGAGAAGCACCATTGGAATAGCCATGCTTGAGAACTAAGCTTG 3421 GCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACAC 3481 AACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTC 3541 ACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTG 3601 CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCT 3661 TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCAC 3721 TCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGA 3781 GCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT 3841 AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAAC 3901 CCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCT 3961 GTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCG 4021 CTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG 4081 GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGT 4141 CTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGG 4201 ATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTAC 4261 GGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGA 4321 AAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTT 4381 GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTT 4441 TCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGA 4501 TTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATC 4561 TAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCT 4621 ATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATA 4681 ACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCA 4741 CGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGA 4801 AGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGA 4861 GTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTG 4921 GTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA 4981 GTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTT 5041 GTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCT 5101 CTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCA 5161 TTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAAT 5221 ACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGA 5281 AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCC 5341 AACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGG 5401 CAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTC 5461 CTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTT 5521 GAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCA 5581 CCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACG 5641 AGGCCCTTTCGTC SEQ IDNO:26(reversePCRprimerintheSV40earlypromoter) 1 AGATGCATGCTTTGCATACTTCTGCCTGC SEQIDNO:27(donorplasmidforinsertingGFPintoFRTInsertiontarget sequence) 1 GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATG 61 CCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCG 121 CGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGC 181 TTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATT 241 GATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA 301 TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC 361 CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCC 421 ATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT 481 ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATT 541 ATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA 601 TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTG 661 ACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACC 721 AAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCG 781 GTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCA 841 CTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGC 901 GTTTAAACTTAAGCTTAGCCACCaTGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGG 961 TGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCG 1021 AGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCA 1081 AGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGAGTGCAGTGCTTCA 1141 GCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCT 1201 ACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGG 1261 TGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGG 1321 AGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA 1381 TCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCG 1441 AGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCC 1501 CCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCA 1561 ACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG 1621 GCATGGACGAGCTGTACAAGTAAGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATA 1681 TCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTC 1741 GACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGAC 1801 CCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTG 1861 TCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGA 1921 TTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGA 1981 AAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGC 2041 GGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGC 2101 TCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCT 2161 AAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAA 2221 ACTTGATTAGGGTGATGGTTCACGTACCTAGAAGTTCCTATTCCGAAGTTCCTATTCTCT 2281 AGAAAGTATAGGAACTTCCTTGGCCAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAA 2341 GTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGA 2401 ATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTG 2461 CGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCC 2521 GATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCG 2581 CCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCA 2641 GCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTT 2701 CGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGC 2761 GATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTC 2821 CGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCA 2881 CCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGC 2941 GGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTT 3001 CTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCA 3061 TCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCA 3121 ACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATG 3181 CGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAG 3241 CGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCC 3301 CAGCACTCGTCCGAGGGCAAAGGAATAGCACGTACTACGAGATTTCGATTCCACCGCCGC 3361 CTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCA 3421 GCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAA 3481 TGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCA 3541 TTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGAC 3601 CTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC 3661 GCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTA 3721 ATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAA 3781 CCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT 3841 TGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCG 3901 AGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC 3961 AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTT 4021 GCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAG 4081 TCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTC 4141 CCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCC 4201 TTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT 4261 CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTT 4321 ATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGC 4381 AGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA 4441 GTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAA 4501 GCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGG 4561 TAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGA 4621 AGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGG 4681 GATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATG 4741 AAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTT 4801 AATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACT 4861 CCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAAT 4921 GATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGG 4981 AAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTG 5041 TTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCAT 5101 TGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTC 5161 CCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTT 5221 CGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGC 5281 AGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA 5341 GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC 5401 GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAA 5461 ACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTA 5521 ACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTG 5581 AGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTG 5641 AATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCAT 5701 GAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATT 5761 TCCCCGAAAAGTGCCACCTGACGTC SEQIDNO:28(reversePCRprimerinthehygromycin-resistancegene) 1 CAGAAACTTCTCGACAGACGTCGCGGTGAG SEQIDNO:29(CHOX-45/46aminoacidsequence) 1 MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSICASIRPEQERKFKHRLVLRFEVTQKTQ 61 RRWFLDKLVDEIGVGYVYDSGSVSRYYLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIE 121 QLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSA 181 ASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSIFATICPRQQYKFKHQL 241 RLRFEVDQKTQRRWFLDKLVDEIGVGYVYDLGSVSRYGLSEIKPLHNFLTQLQPFLKLKQ 301 KQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKK 361 KSSP SEQIDNO:30(CHOX-45/46recognitionsitesequence) 1 CAGCACGTCTCACCCCACCCCT SEQIDNO:31(CHOX-45/46forwardscreeningprimer) 1 GGAATCTGACTGTGGTAAGCCTGTACAC SEQIDNO:32(CHOX-45/46reversescreeningprimer) 1 CAGCACTCAGGAGGTAGAGGCAGG SEQIDNO:33(artificialspliceacceptor) 1 TCTTACTGACATCCACTTTGCCTTTCTCTCCACAGG SEQIDNO:34(SV40polyadenylationsignal) 1 ACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAA 61 ATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTT 121 ATCATGTCTG SEQIDNO:35(BGHpolyadenylationsignal) 1 CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCC 61 TGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTC 121 TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATT 181 GGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG