US20040067225A1

US20040067225A1 - Isolated nucleic acid molecules human glucuronyltransferase proteins, and related products and processes

Info

Publication number: US20040067225A1
Application number: US10/634,905
Authority: US
Inventors: Weiniu Gan; Chunhua Yan; Gennady Merkulov; Valentina Di Francesco; Ellen Beasley
Original assignee: Applera Corp
Current assignee: Applied Biosystems Inc
Priority date: 2001-02-26
Filing date: 2003-08-06
Publication date: 2004-04-08
Also published as: AU2002240295A1; WO2002068657A3; US6664084B2; WO2002068657A2; US20020137164A1; CA2439283A1; EP1368479A2

Abstract

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the enzyme peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the enzyme peptides, and methods of identifying modulators of the enzyme peptides.

Description

FIELD OF THE INVENTION

The present invention is in the field of enzyme proteins that are related to the transferase enzyme subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

Many human enzymes serve as targets for the action of pharmaceutically active compounds. Several classes of human enzymes that serve as such targets include helicase, steroid esterase and sulfatase, convertase, synthase, dehydrogenase, monoxygenase, transferase, kinase, glutanase, decarboxylase, isomerase and reductase. It is therefore important in developing new pharmaceutical compounds to identify target enzyme proteins that can be put into high-throughput screening formats. The present invention advances the state of the art by providing novel human drug target enzymes related to the transferase subfamily.

Transferases

The novel human protein, and encoding gene, provided by the present invention shows the greatest degree of similarity to a glucuronyltransferase cloned from a rat brain cDNA library (Seiki et al., Biochem. Biophys. Res. Commun. 255 (1), 182-187 (1999)). This glucuronyltransferase is important for the biosynthesis of the HNK-1 carbohydrate epitope of glycoproteins. It was demonstrated that transfection of glucuronyltransferase cDNA into COS-1 cells caused the HNK-1 carbohydrate epitope to be expressed on the cell surfaces and also caused the cells to undergo morphological changes. The rat glucuronyltransferase has type II transmembrane topology and the amino acid sequence shares 49% identity with rat GlcAT-P, which is another glucuronyltransferase involved in HNK-1 biosynthesis (Seiki et al., Biochem. Biophys. Res. Commun. 255 (1), 182-187 (1999); for information on GlcAT-P, see Terayama et al. (1997) Proc. Natl. Acad. Sci. USA 94, 6093-6098).

Enzyme proteins, particularly members of the transferase enzyme subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of enzyme proteins. The present invention advances the state of the art by providing previously unidentified human enzyme proteins, and the polynucleotides encoding them, that have homology to members of the transferase enzyme subfamily. These novel compositions are useful in the diagnosis, prevention and treatment of biological processes associated with human diseases.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of amino acid sequences of human enzyme peptides and proteins that are related to the transferase enzyme subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate enzyme activity in cells and tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodes the enzyme protein of the present invention. (SEQ ID NO:1) In addition, structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. [0007]
FIG. 2 provides the predicted amino acid sequence of the enzyme of the present invention. (SEQ ID NO:2) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. [0008]
FIG. 3 provides genomic sequences that span the gene encoding the enzyme protein of the present invention. (SEQ ID NO:3) In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. As illustrated in FIG. 3, SNPs were identified at 35 different nucleotide positions.[0009]

DETAILED DESCRIPTION OF THE INVENTION

General Description [0010]
The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a enzyme protein or part of a enzyme protein and are related to the transferase enzyme subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human enzyme peptides and proteins that are related to the transferase enzyme subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these enzyme peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the enzyme of the present invention. [0011]
In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known enzyme proteins of the transferase enzyme subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known transferase family or subfamily of enzyme proteins. [0012]
Specific Embodiments [0013]
Peptide Molecules [0014]
The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the enzyme family of proteins and are related to the transferase enzyme subfamily (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the enzyme peptides of the present invention, enzyme peptides, or peptides/proteins of the present invention. [0015]
The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprise the amino acid sequences of the enzyme peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below. [0016]
As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below). [0017]
In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation. [0018]
The language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the enzyme peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals. [0019]
The isolated enzyme peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. For example, a nucleic acid molecule encoding the enzyme peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below. [0020]
Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein. [0021]
The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein. [0022]
The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the enzyme peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below. [0023]
The enzyme peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a enzyme peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the enzyme peptide. “Operatively linked” indicates that the enzyme peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the enzyme peptide. [0024]
In some uses, the fusion protein does not affect the activity of the enzyme peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant enzyme peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence. [0025]
A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., [0026] Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A enzyme peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the enzyme peptide.
As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention. [0027]
Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the enzyme peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs. [0028]
To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. [0029]
The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. ([0030] Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. ([0031] J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the enzyme peptides of the present invention as well as being encoded by the same genetic locus as the enzyme peptide provided herein. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 6 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. [0032]
Allelic variants of a enzyme peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the enzyme peptide as well as being encoded by the same genetic locus as the enzyme peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 6 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a enzyme peptide encoding nucleic acid molecule under stringent conditions as more fully described below. [0033]
FIG. 3 provides information on SNPs that have been found in the gene encoding the enzyme of the present invention. SNPs were identified at 35 different nucleotide positions, including a non-synonymous coding SNP at nucleotide position 96648 (protein position 261). The change in the amino acid sequence caused by this SNP is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. Some of these SNPs that are located outside the ORF and in introns may affect gene transcription. [0034]
Paralogs of a enzyme peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the enzyme peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a enzyme peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below. [0035]
Orthologs of a enzyme peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the enzyme peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a enzyme peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins. [0036]
Non-naturally occurring variants of the enzyme peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the enzyme peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a enzyme peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., [0037] Science 247:1306-1310 (1990).
Variant enzyme peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to phosphorylate substrate, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree. [0038]
Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region. [0039]
Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., [0040] Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as enzyme activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).
The present invention further provides fragments of the enzyme peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention. [0041]
As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a enzyme peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the enzyme peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the enzyme peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2. [0042]
Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in enzyme peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2). [0043]
Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. [0044]
Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as [0045] Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
Accordingly, the enzyme peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature enzyme peptide is fused with another compound, such as a compound to increase the half-life of the enzyme peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature enzyme peptide, such as a leader or secretory sequence or a sequence for purification of the mature enzyme peptide or a pro-protein sequence. [0046]
Protein/Peptide Uses [0047]
The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a enzyme-effector protein interaction or enzyme-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products. [0048]
Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987. [0049]
The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, enzymes isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the enzyme. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in testis, prostate, kidney, brain, ear, and lung, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. A large percentage of pharmaceutical agents are being developed that modulate the activity of enzyme proteins, particularly members of the transferase subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. Such uses can readily be determined using the information provided herein, that which is known in the art, and routine experimentation. [0050]
The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to enzymes that are related to members of the transferase subfamily. Such assays involve any of the known enzyme functions or activities or properties useful for diagnosis and treatment of enzyme-related conditions that are specific for the subfamily of enzymes that the one of the present invention belongs to, particularly in cells and tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in testis, prostate, kidney, brain, ear, and lung, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. [0051]
The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems. Cell-based systems can be native, i.e., cells that normally express the enzyme, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the enzyme protein. [0052]
The polypeptides can be used to identify compounds that modulate enzyme activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the enzyme. Both the enzymes of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the enzyme. These compounds can be further screened against a functional enzyme to determine the effect of the compound on the enzyme activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the enzyme to a desired degree. [0053]
Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the enzyme protein and a molecule that normally interacts with the enzyme protein, e.g. a substrate or a component of the signal pathway that the enzyme protein normally interacts (for example, another enzyme). Such assays typically include the steps of combining the enzyme protein with a candidate compound under conditions that allow the enzyme protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the enzyme protein and the target, such as any of the associated effects of signal transduction such as protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc. [0054]
Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., [0055] Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).
One candidate compound is a soluble fragment of the receptor that competes for substrate binding. Other candidate compounds include mutant enzymes or appropriate fragments containing mutations that affect enzyme function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention. [0056]
The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) enzyme activity. The assays typically involve an assay of events in the signal transduction pathway that indicate enzyme activity. Thus, the phosphorylation of a substrate, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the enzyme protein dependent signal cascade can be assayed. [0057]
Any of the biological or biochemical functions mediated by the enzyme can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the enzyme can be assayed. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in testis, prostate, kidney, brain, ear, and lung, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. [0058]
Binding and/or activating compounds can also be screened by using chimeric enzyme proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a substrate-binding region can be used that interacts with a different substrate then that which is recognized by the native enzyme. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the enzyme is derived. [0059]
The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the enzyme (e.g. binding partners and/or ligands). Thus, a compound is exposed to a enzyme polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble enzyme polypeptide is also added to the mixture. If the test compound interacts with the soluble enzyme polypeptide, it decreases the amount of complex formed or activity from the enzyme target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the enzyme. Thus, the soluble polypeptide that competes with the target enzyme region is designed to contain peptide sequences corresponding to the region of interest. [0060]
To perform cell free drug screening assays, it is sometimes desirable to immobilize either the enzyme protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. [0061]
Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., [0062] ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of enzyme-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a enzyme-binding protein and a candidate compound are incubated in the enzyme protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the enzyme protein target molecule, or which are reactive with enzyme protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
Agents that modulate one of the enzymes of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context. [0063]
Modulators of enzyme protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the enzyme pathway, by treating cells or tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. These methods of treatment include the steps of administering a modulator of enzyme activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein. [0064]
In yet another aspect of the invention, the enzyme proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) [0065] Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the enzyme and are involved in enzyme activity. Such enzyme-binding proteins are also likely to be involved in the propagation of signals by the enzyme proteins or enzyme targets as, for example, downstream elements of a enzyme-mediated signaling pathway. Alternatively, such enzyme-binding proteins are likely to be enzyme inhibitors.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a enzyme protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a enzyme-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the enzyme protein. [0066]
This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a enzyme-modulating agent, an antisense enzyme nucleic acid molecule, a enzyme-specific antibody, or a enzyme-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. [0067]
The enzyme proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. The method involves contacting a biological sample with a compound capable of interacting with the enzyme protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array. [0068]
One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. [0069]
The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered enzyme activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array. [0070]
In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample. [0071]
The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. ([0072] Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the enzyme protein in which one or more of the enzyme functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other substrate-binding regions that are more or less active in substrate binding, and enzyme activation. Accordingly, substrate dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.
The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. Accordingly, methods for treatment include the use of the enzyme protein or fragments. [0073]
Antibodies [0074]
The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity. [0075]
As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′)[0076] ₂, and Fv fragments.
Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989). [0077]
In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures. [0078]
Antibodies are preferably prepared from regions or discrete fragments of the enzyme proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or enzyme/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments. [0079]
An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2). [0080]
Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include [0081] ¹²⁵I, ¹³¹I, ³⁵S or ³H.
Antibody Uses [0082]
The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in testis, prostate, kidney, brain, ear, and lung, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover. [0083]
Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein. [0084]
The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy. [0085]
Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art. [0086]
The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type. [0087]
The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the enzyme peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention. [0088]
The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nuleic acid arrays and similar methods have been developed for antibody arrays. [0089]
Nucleic Acid Molecules [0090]
The present invention further provides isolated nucleic acid molecules that encode a enzyme peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the enzyme peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof. [0091]
As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5KB, 4KB, 3KB, 2KB, or 1KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences. [0092]
Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated. [0093]
For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically. [0094]
Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIG. 1 or [0095] 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.
The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIG. 1 or [0096] 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.
The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIG. 1 or [0097] 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.
In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein. [0098]
The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes. [0099]
As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the enzyme peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification. [0100]
Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand). [0101]
The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the enzyme proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions. [0102]
The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3. [0103]
A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene. [0104]
A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides. [0105]
Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 6 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. [0106]
FIG. 3 provides information on SNPs that have been found in the gene encoding the enzyme of the present invention. SNPs were identified at 35 different nucleotide positions, including a non-synonymous coding SNP at nucleotide position 96648 (protein position 261). The change in the amino acid sequence caused by this SNP is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. Some of these SNPs that are located outside the ORF and in introns may affect gene transcription. [0107]
As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in [0108] Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to low stringency hybridization conditions are well known in the art.
Nucleic Acid Molecule Uses [0109]
The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2. As illustrated in FIG. 3, SNPs were identified at 35 different nucleotide positions. [0110]
The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention. [0111]
The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence. [0112]
The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations. [0113]
The nucleic acid molecules are also useful for expressing antigenic portions of the proteins. [0114]
The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 6 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. [0115]
The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention. [0116]
The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein. [0117]
The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides. [0118]
The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides. [0119]
The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides. [0120]
The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in testis, prostate, kidney, brain, ear, and lung, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in enzyme protein expression relative to normal results. [0121]
In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization. [0122]
Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a enzyme protein, such as by measuring a level of a enzyme-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a enzyme gene has been mutated. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in testis, prostate, kidney, brain, ear, and lung, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. [0123]
Nucleic acid expression assays are useful for drug screening to identify compounds that modulate enzyme nucleic acid expression. [0124]
The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the enzyme gene, particularly biological and pathological processes that are mediated by the enzyme in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. The method typically includes assaying the ability of the compound to modulate the expression of the enzyme nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired enzyme nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the enzyme nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences. [0125]
The assay for enzyme nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the enzyme protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase. [0126]
Thus, modulators of enzyme gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of enzyme mRNA in the presence of the candidate compound is compared to the level of expression of enzyme mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression. [0127]
The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate enzyme nucleic acid expression in cells and tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in testis, prostate, kidney, brain, ear, and lung, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression. [0128]
Alternatively, a modulator for enzyme nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the enzyme nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in humans in testis, prostate, kidney, brain (including fetal brain), ear, and lung. [0129]
The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the enzyme gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased. [0130]
The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in enzyme nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in enzyme genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the enzyme gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the enzyme gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a enzyme protein. [0131]
Individuals carrying mutations in the enzyme gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been found in the gene encoding the enzyme of the present invention. SNPs were identified at 35 different nucleotide positions, including a non-synonymous coding SNP at nucleotide position 96648 (protein position 261). The change in the amino acid sequence caused by this SNP is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. Some of these SNPs that are located outside the ORF and in introns may affect gene transcription. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 6 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., [0132] Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.
Alternatively, mutations in a enzyme gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis. [0133]
Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature. [0134]
Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant enzyme gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) [0135] Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).
Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., [0136] Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.
The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the enzyme gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been found in the gene encoding the enzyme of the present invention. SNPs were identified at 35 different nucleotide positions, including a non-synonymous coding SNP at nucleotide position 96648 (protein position 261). The change in the amino acid sequence caused by this SNP is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. Some of these SNPs that are located outside the ORF and in introns may affect gene transcription. [0137]
Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens. [0138]
The nucleic acid molecules are thus useful as antisense constructs to control enzyme gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of enzyme protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into enzyme protein. [0139]
Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of enzyme nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired enzyme nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the enzyme protein, such as substrate binding. [0140]
The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in enzyme gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired enzyme protein to treat the individual. [0141]
The invention also encompasses kits for detecting the presence of a enzyme nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in testis, prostate, kidney, brain, ear, and lung, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting enzyme nucleic acid in a biological sample; means for determining the amount of enzyme nucleic acid in the sample; and means for comparing the amount of enzyme nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect enzyme protein mRNA or DNA. [0142]
Nucleic Acid Arrays [0143]
The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3). [0144]
As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522. [0145]
The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest. [0146]
In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support. [0147]
In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation. [0148]
In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples. [0149]
Using such arrays, the present invention provides methods to identify the expression of the enzyme proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the enzyme gene of the present invention. FIG. 3 provides information on SNPs that have been found in the gene encoding the enzyme of the present invention. SNPs were identified at 35 different nucleotide positions, including a non-synonymous coding SNP at nucleotide position 96648 (protein position 261). The change in the amino acid sequence caused by this SNP is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. Some of these SNPs that are located outside the ORF and in introns may affect gene transcription. [0150]
Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, [0151] An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized. [0152]
In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention. [0153]
Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid. [0154]
In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified enzyme gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays. [0155]
Vectors/Host Cells [0156]
The invention also provides vectors containing the nucleic acid molecules described herein. The term “vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC. [0157]
A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates. [0158]
The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors). [0159]
Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system. [0160]
The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from [0161] E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.
In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers. [0162]
In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., [0163] Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).
A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., [0164] Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).
The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art. [0165]
The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art. [0166]
The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, [0167] E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.
As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enteroenzyme. Typical fusion expression vectors include pGEX (Smith et al., [0168] Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).
Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., [0169] Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).
The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., [0170] S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., [0171] Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).
In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. [0172] Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).
The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. [0173] Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression). [0174]
The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells. [0175]
The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. ([0176] Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector. [0177]
In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects. [0178]
Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective. [0179]
While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein. [0180]
Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as enzymes, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides. [0181]
Where the peptide is not secreted into the medium, which is typically the case with enzymes, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography. [0182]
It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process. [0183]
Uses of Vectors and Host Cells [0184]
The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a enzyme protein or peptide that can be further purified to produce desired amounts of enzyme protein or fragments. Thus, host cells containing expression vectors are useful for peptide production. [0185]
Host cells are also useful for conducting cell-based assays involving the enzyme protein or enzyme protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native enzyme protein is useful for assaying compounds that stimulate or inhibit enzyme protein function. [0186]
Host cells are also useful for identifying enzyme protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant enzyme protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native enzyme protein. [0187]
Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a enzyme protein and identifying and evaluating modulators of enzyme protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians. [0188]
A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the enzyme protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse. [0189]
Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the enzyme protein to particular cells. [0190]
Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., [0191] Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.
In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. [0192] PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. [0193] Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G_ophase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect substrate binding, enzyme protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo enzyme protein function, including substrate interaction, the effect of specific mutant enzyme proteins on enzyme protein function and substrate interaction, and the effect of chimeric enzyme proteins. It is also possible to assess the effect of null mutations, that is, mutations that substantially or completely eliminate one or more enzyme protein function. [0194]
All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims. [0195]
1 5 1 4970 DNA Homo sapiens 1 gttctgagaa gacaagagca agggactgag agcaggcttc cgctgcggcg cgcgaacaca 60 gccgggacac aacccccagc gtctccaccc gctcctcgcc accccggcgg gaatgtgagg 120 aaggaaagcc cccagcgccg ccgcccgccc tcgaaggcgt cccagagagc gtcctggggg 180 cccgcggctg gagcccttgt gcccgcagca ccgccggact ggagcggcga ggcgcaccgg 240 gtgccgcttc tcggcttcca ctcttcagaa agagcgcggt ggggatcagc gcctttcccg 300 cactcggcac aactccggga ccggcggcgc gcggctggac cgagtcccgc ttcccgccag 360 ctcacctgga gtcgggggca gcccctgccc gcccgcctgc accccttgtc gctctagctt 420 gcgcgaacct gccgctcctc cacgcccagg tagtgagccc cgcggctcca ggtctctgca 480 gcgccctcgg ccccatggac agcgcaccca tcaccactcc ctaagtgctg gcgccgccgc 540 tgtccaagct gcgcactggg gtccctcggc tcgcccctct ctggggtgtc cgagaggcca 600 gggagcgtgc accatgaagt ccgcgctttt cacccgcttc tttatcctcc tgccctggat 660 cctaattgtc atcatcatgc tcgacgtgga cacgcgcagg ccagtgcccc cgctcacccc 720 gcgcccctac ttctctccct acgcggtggg ccgcgggggc gcccgactcc cgctccgcag 780 gggcggcccg gctcacggga cccaaaagcg caaccagtct cggccgcagc cacagccgga 840 gccgcagctg cccaccatct atgccatcac gcccacctac agccgcccgg tgcagaaagc 900 ggagctgacc cgcctggcca acacgttccg ccaggtggcg cagctgcact ggatcctggt 960 ggaggacgcg gcggcgcgca gcgagctggt gagccgcttc ctggcgcggg ccgggctgcc 1020 cagcactcac ctgcacgtgc ccacgccgcg gcgctacaag cggcccgggc tgccgcgcgc 1080 cactgagcag cgcaacgcgg gcctcgcctg gctgcgccag aggcaccagc accagcgcgc 1140 gcagcccggc gtgctcttct tcgctgacga cgacaacacc tatagtctgg agctcttcca 1200 ggagatgcga accacccgca aggtctccgt ctggcctgtg ggcctggttg gtgggcggcg 1260 ctacgaacgt ccgctggtgg aaaacggcaa agttgttggc tggtacaccg gctggagagc 1320 agacaggcct tttgccatcg acatggcagg atttgctgta agtcttcaag tcattttgtc 1380 caatccaaaa gctgtattta agcgtcgtgg atcccagcca gggatgcaag aatctgactt 1440 tctcaaacag ataacaacag tcgaagaact ggaaccgaaa gcaaataact gcactaaggt 1500 tctcgtgtgg cacactcgga cagagaaggt taatctagcc aacgagccaa agtaccacct 1560 ggacacagtg aaaattgagg tataataaat tgaagcagca actggtgcag tttgtccagc 1620 cagtggatcc atatggaaga ggatgtttgg agtttaggct acagagcatt caggtattgt 1680 ttgttttact tcagtacagc agcctttctt gtcatctgat ggacatctgt ttaaatggag 1740 cttgtcagtt aacataagct aattggatgg ttggtacaaa atgtatgttt tgtcttcatt 1800 tgttctgcat gttttctcta caacaactaa attggaagat ttttttgtac agtgccgata 1860 ctgcaagata ccactcttga gtatatattt tttctttttc tccaatttgc ccttataatt 1920 ggtagacttg aacaggttgg tagacttgaa caggttttta aaacagacaa gtattttgtc 1980 agctaaacgt tcctgatgat tcctgacttt gcaatactaa gtaatttttg gaaggttagt 2040 ggcagtatac atcataggaa ataaaaaccc acaaatgaaa aggtctatgg agtcatgttt 2100 aatgtaggga aataacattt tgtcaatact aggcaccata aaatgtaaac acaattactg 2160 tcataaacct agatatacct tcaaggattg aagattgaaa gtggctttgt tttagttagt 2220 taccctgttt gcatatagtg cagaaaaagg tcttcatgtt agcactatgt acattaagaa 2280 gagatccaaa ttacaagaga ggcagataaa atttgaattc tttaagcatt cattaaacga 2340 agttttggag taacatccac gtttatcttc ctttcactaa tcacgttccc tgttaagcac 2400 atcataacaa cagcacagtg aagtgaatga tgaaataaga gcattttgat acactagaaa 2460 acagtgctca gtgagacatt tacattctat ttatatgatt aaacatttga tcatacagta 2520 ccttcctaca ggattactgg ctaattttgg ggtggggttt atactattag aggtattact 2580 aacatgataa ctacttccct tatatgcaaa cattagagct ataattttat tgagaggaaa 2640 actgattttg caagttgagc agcttctcaa ataatgcagt acatgaaatc atgggaaata 2700 tgagcaaagc tgcccttgac ataaaatgat ttatcaacct gcttttcacc acatcaaatt 2760 gaatcagtac agaccaacac ggtcaatcag atcattctta atatgaacaa atgggtaaaa 2820 agaaaaaaaa tatgcatatg aataaacagg ggaactagat gcgtttcagc aaggaatgtc 2880 aggtggtagt tctggatgaa acttgtattg cagttttcat ttccacagtt gtgtgctgag 2940 agtctgacct gatgagcttc cagaccatcc tgctgttgtg ctggagggct ggccaaaacc 3000 tgcagtaggg gttgcactac tgatactcat gccagccatc tgctgattca tctgtgaaac 3060 atataaaagg cttagttcaa gaggcttact tcacttttaa ttcttgtttc tttagccaca 3120 cagttggtca ttttttcatt aatgtgacaa ctagtccaag cactggaata aaaacagagt 3180 accatacaaa tatttcttaa agcaaatagc tactttgttc ccttctttat ctactttcta 3240 gatacagttt ccccaaagat taaccacaac ttacttaaaa aaaaatacca aagcaatctt 3300 gggattttaa tgagtccgct actctaacta actttcacct acactaggat attgtgcttt 3360 aactactaag gagtaagaaa attttaggaa gtaaaatagt ctaaaattat cctataaact 3420 ttgtatgata gatattattc tctattaaaa tcttatatac ttcctaaata tttttaaagt 3480 ggtcataaag catttatttc tctcgctgat ctaacaacat aaacatctaa aatttatttt 3540 cattgtatgc aataaagcat aagattacat gtatttttct tcaagactgg agtcaaatat 3600 atatatatat aagcatctta accctgtgat tctcttactt ccaaaattgg tgataagaga 3660 aggaaaggca agatttacca tatagtgagt gggtttaaaa cttacactca gagttagact 3720 gtgttcttaa tttaatacat ttgacttgac ttatttacag tttcaaagac actaacataa 3780 actacatcac taatcaggca taagtgtctg aagaagcaga tcacatcttc atacctacta 3840 aaggacattt taaccacctt gtcattggcc agtagattgc actgatggag tgctggagaa 3900 cagcatcacc cttctgcatt atctggaagt aagagccagt attaactcct tcctggttca 3960 tctagcacct taacctgagc tgggtgtgct tcagcatgtt gaccatgtga ctgacactta 4020 gcacatacaa ttttttagat tcccagcggg tagagaccaa tgttttacct atattcttgt 4080 aaatggtggt agcaaaatta actgtgatat atagtgattg tgctaatgtt agaaatcact 4140 ctagactatt ccctgaatgc tctaaaggta aaacaagtga ccaaacagaa accaagattg 4200 ccaaaatgct ggaggaacat caatgggaag tgtaaaagga agaagagtgg gagcatgaac 4260 ctctctaaga gcctttgtct gtgcagctag agaaaagtca gaacacagca cctgaaatag 4320 aaatgttcta tctcagctct aacttaggta gaaataggat tttataatat gaggggatgt 4380 ctggttcaca ccttatggga attgaatctt tttgtactct ttttaaacat aaaagtcatt 4440 atagggtatg taaaaagaaa atacaacttt acaaaggttt ctcaacaaaa agaattttta 4500 cagagccatg gggcagtaat catccgacct gaaaaatagc cttagatccc tcataaaata 4560 gtgctttgag aatatgaggc tagattttta ttttctaata aaagatccta aaattattag 4620 tgaagctaag ttgtctaagt ggactgtaaa aatgtcccac cagcaagctg gataaagctt 4680 agtgctaatc tcagaggtga cagagaggga gtctcatgat gcctgagata atttctggcc 4740 attagtggtg ttcacgttac agttacatta caatttgaaa tgaaagatgt ttaacctttt 4800 ttttacagaa tactctaaaa tagggataat aacacatcat ttgtctatct gatacaactt 4860 cataatattt atagatactt ttgaccctat aacatattat ccctattgaa ttctttctgc 4920 cagatcacta gacttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4970 2 323 PRT Homo sapiens 2 Met Lys Ser Ala Leu Phe Thr Arg Phe Phe Ile Leu Leu Pro Trp Ile 1 5 10 15 Leu Ile Val Ile Ile Met Leu Asp Val Asp Thr Arg Arg Pro Val Pro 20 25 30 Pro Leu Thr Pro Arg Pro Tyr Phe Ser Pro Tyr Ala Val Gly Arg Gly 35 40 45 Gly Ala Arg Leu Pro Leu Arg Arg Gly Gly Pro Ala His Gly Thr Gln 50 55 60 Lys Arg Asn Gln Ser Arg Pro Gln Pro Gln Pro Glu Pro Gln Leu Pro 65 70 75 80 Thr Ile Tyr Ala Ile Thr Pro Thr Tyr Ser Arg Pro Val Gln Lys Ala 85 90 95 Glu Leu Thr Arg Leu Ala Asn Thr Phe Arg Gln Val Ala Gln Leu His 100 105 110 Trp Ile Leu Val Glu Asp Ala Ala Ala Arg Ser Glu Leu Val Ser Arg 115 120 125 Phe Leu Ala Arg Ala Gly Leu Pro Ser Thr His Leu His Val Pro Thr 130 135 140 Pro Arg Arg Tyr Lys Arg Pro Gly Leu Pro Arg Ala Thr Glu Gln Arg 145 150 155 160 Asn Ala Gly Leu Ala Trp Leu Arg Gln Arg His Gln His Gln Arg Ala 165 170 175 Gln Pro Gly Val Leu Phe Phe Ala Asp Asp Asp Asn Thr Tyr Ser Leu 180 185 190 Glu Leu Phe Gln Glu Met Arg Thr Thr Arg Lys Val Ser Val Trp Pro 195 200 205 Val Gly Leu Val Gly Gly Arg Arg Tyr Glu Arg Pro Leu Val Glu Asn 210 215 220 Gly Lys Val Val Gly Trp Tyr Thr Gly Trp Arg Ala Asp Arg Pro Phe 225 230 235 240 Ala Ile Asp Met Ala Gly Phe Ala Val Ser Leu Gln Val Ile Leu Ser 245 250 255 Asn Pro Lys Ala Val Phe Lys Arg Arg Gly Ser Gln Pro Gly Met Gln 260 265 270 Glu Ser Asp Phe Leu Lys Gln Ile Thr Thr Val Glu Glu Leu Glu Pro 275 280 285 Lys Ala Asn Asn Cys Thr Lys Val Leu Val Trp His Thr Arg Thr Glu 290 295 300 Lys Val Asn Leu Ala Asn Glu Pro Lys Tyr His Leu Asp Thr Val Lys 305 310 315 320 Ile Glu Val 3 99916 DNA Homo sapiens misc_feature (1)...(99916) n = A,T,C or G 3 cacagtgttt catctctgaa tagccttggt tcctggagag gatgtaatag caaataggtt 60 ttatggcata gttggaccaa ccaaaagttc cccactaaca aaggttagtg atgtttttag 120 tacaattcag taacaacatg aattttattc cagcaaacac aaggcatttt aaccaaattt 180 aaaatttcca acaggatgaa ccagacttca gtaacttagg ggagaggggt gcactgcttc 240 ctaagaaaaa ccttttctta aagggaggcc ttgacttctt tcctcttccc catcctggca 300 tgatgctggt gcagaataca tttccaaggt tgtgttatat tcagaggcac attaggctat 360 tcgagagaat gggaagaggt ctaatggctt cgttgttttc tatgtttagg atgcactaat 420 ctgcagagtt taatgtatga gttgtggatg gcacagggaa gttgggacag aaggctagct 480 ctagggagtt gttggggtgg ctgggtgtta gtctgaaatt tcactctcac gtttcagaat 540 ctgaagagga agttagggaa attcacagat gagccaatgc cttttaacag gtggggggtt 600 caggggtagg actgggagaa gaggagctgt cagaaaagct ggagagccat tcgccccaga 660 gctctcagtt gcaccagaac gcacagtcgg agagagattt atttgtgaac aagcccgaga 720 aaggcagaca acccagacaa gatgggctca gaaattattt atcataggct agggcagaac 780 agtctggagc tttccttctg ctttgcaagc acttccctgg gacctgcctg ggaagcacag 840 cccttcttcc tggtaagtta cagaaggtca ggcactcagc tagtaggcca gctagacaac 900 aaaaagtgtc taaactaagt gccaactgtt tacttggggt cttccagacc ctatcatggt 960 tctattcgtc atggtacact ctgtttgatt tcaatcgcac catctttata tgtaggaagg 1020 ccaaaccggc ccggctgtgt tgggtgggat ttctgcaggc catgcagtga ggacgccact 1080 agctatagac acacgcagga gacccacgtt ctatttccat atgccacttg ccactgacct 1140 cagagaaatc attttaaacc ctgaacctca gcttccttat cctgaaaatg aaggccattc 1200 tacttcttag gccttcttga caggagcctt gggagagcca aacggagtaa ttccggtgac 1260 agcgaagaac tgggagggac cgtgactcca aattactgaa gaaaaataag ttaaacatgc 1320 tcggctggag ttttaggaat tccacccgcc cccacccgcc atgatcaaac tgcagtcagg 1380 cagcagccct ccgaattgat tctttcttat ggactgtctc acatgccaaa cacctatgtg 1440 cagcacccct ccatctccgt gggctcctat tgatgtctgc cttgcagtga aagcgctttt 1500 tgttgccgct gttcgataca ccccagcaca tcaacgcagc agctgggagc aggaggagga 1560 atcccttcgc tttctcccct cctttcactc cgaaccctag gcatcagcag caacgcgcgt 1620 agcccggcaa atttccgcac gtgaatggat tttcctggct catctccaaa gaccttgcgg 1680 tatttcacaa agctgaagag aggtgaacga gcatcacccc ctccaggagg tcgtgcagcc 1740 ccgcggggac gctcctcccg ctcctccacc cgcggtgcag cggccgcctc ccgagcttgc 1800 tcgcggggcc gttagcgcca gacccggcgg agagccaagg ggtccctccg cgcctccctc 1860 tcgcagcccc tctccaccag tagcagcgct gctgtcttcc cacaggagga cttgggagga 1920 cgctggattc tgcagcgccc ccgccccctc gctttttctt tctttccttg ctttgggatc 1980 ttgctgctgg atccggagag gttctgagaa gacaagagca agggactgag agcaggcttc 2040 cgctgcggcg cgcgaacaca gccgggacac aacccccagc gtctccaccc gctcctcgcc 2100 accccggcgg gaatgtgagg aaggaaagcc cccagcgccg ccgcccgccc tcgaaggcgt 2160 cccagagagc gtcctggggg cccgcggctg gagcccttgt gcccgcagca ccgccggact 2220 ggagcggcga ggcgcaccgg gtgccgcttc tcggcttcca ctcttcagaa agagcgcggt 2280 ggggatcagc gcctttcccg cactcggcac aactccggga ccggcggcgc gcggctggac 2340 cgagtcccgc ttcccgccag ctcacctgga gtcgggggca gcccctgccc gcccgcctgc 2400 accccttgtc gctctagctt gcgcgaacct gccgctcctc cacgcccagg tagtgagccc 2460 cgcggctcca ggtctctgca gcgccctcgg ccccatggac agcgcaccca tcaccactcc 2520 ctaagtgctg gcgccgccgc tgtccaagct gcgcactggg gtccctcggc tcgcccctct 2580 ctggggtgtc cgagaggcca gggagcgtgc accatgaagt ccgcgctttt cacccgcttc 2640 tttatcctcc tgccctggat cctaattgtc atcatcatgc tcgacgtgga cacgcgcagg 2700 ccagtgcccc cgctcacccc gcgcccctac ttctctccct acgcggtggg ccgcgggggc 2760 gcccgactcc cgctccgcag gggcggcccg gctcacggga cccaaaagcg caaccagtct 2820 cggccgcagc cacagccgga gccgcagctg cccaccatct atgccatcac gcccacctac 2880 agccgcccgg tgcagaaagc ggagctgacc cgcctggcca acacgttccg ccaggtggcg 2940 cagctgcact ggatcctggt ggaggacgcg gcggcgcgca gcgagctggt gagccgcttc 3000 ctggcgcggg ccgggctgcc cagcactcac ctgcacgtgc ccacgccgcg gcgctacaag 3060 cggcccgggc tgccgcgcgc cactgagcag cgcaacgcgg gcctcgcctg gctgcgccag 3120 aggcaccagc accagcgcgc gcagcccggc gtgctcttct tcgctgacga cgacaacacc 3180 tatagtctgg agctcttcca ggaggtaaag gccagcctgc cccgctgggt gggcgagggc 3240 gggagtgggc ctccgggccg gccgggctgc agtcacacgc cccttgcact ccgggtgcac 3300 ttttgagttc tcagttctcc gtgcgcgcat tcggggcacc gagtggagcc gctccttgct 3360 ggcactccgc agcctccgct ggccgtgggg gtggaggggc tgtgtgtgag aggatcactg 3420 tggcttaagg ggcgggagtc tgccctgggg cttttctgtg tggagattgt gtcaagagaa 3480 tagcacaggt gtgaggcgcg ggaatgattt ccaggggcca ggctcctgac gacctgagga 3540 tggagcttag acctgcaggc gctggctgcg ataggagatc agggagggac ctgcaccgta 3600 gcgggtggcg gtggaggggt gggggtggtg gggacgggta ggctaggtgt tactcgaggc 3660 ttttcagtgc ctacaggtgt gattctcagt ctaggaccat atagggagaa aggtggagat 3720 agcctagaga gaaatttgac tattgcggcc cactggcacg gaaatggttg ttagaagaaa 3780 cactgacttc tatggaggtc cttcctctct ttcccacacc tcacaaaagt cttttctgac 3840 cctcaagatt actttttttt tttttttgca agaaaaccct ttctggaaaa taaactgttc 3900 tgccctgtgg ttacgtcttt tttgcagccc aaggtaaaca tctgggctgc ctgatccctt 3960 ttcacaacct ttctctaagg cctctttttg gcgagttagc caggacacat ttggcggggc 4020 cttttgcgtc tttctggtgc agccaggtcc tgagtgcatc cgggctccct gctgttgaga 4080 tggcccatga gttccctggg atggccacgc gcgcaccgcc cccccccgcc gcccccggca 4140 ggcacagggt tagaacaggt gttcttcctt gaggctggta tcttgcggtg tgtgtgtgtg 4200 tagcctgcaa atgagactga gtggcatggg ttttctcagt tttcttctgt ctacgcaatt 4260 acagccaaag aaaatcttct tgattgattt gacgccctgt gagactgttg ccttcctcca 4320 cctgaaattt cttgacgtcc tgcttcagag actccccctc aattcccctt ctcccaagta 4380 aactggacat tgggaaaata ctatgtgtga gttggacact gaactgctaa aataatctgt 4440 gtgtctggtc atcagatacc ctaactgctt cccacacttt catcctcatc tctgggttgc 4500 tttcttaacc ccagggcaga gctattctgt actcttccag tttaattttt ctgtgtggct 4560 ttaaaaaaat tttcttaaag gtaattgccg ccttcatttc tattcatcct ctctttcatg 4620 aataaaaggc acagtaattg tccttcatct tttcttttcc atcctttcac tgtagagggc 4680 tttctttcca cgtttctcca aatgaataaa taagcttggc attgtatagt atggtcctta 4740 ctcaacccaa gattagagat accatctgac gtttttaact gggcttttaa tcctgcatca 4800 gtgctccccc ctccaaaaga aaaaagtggc ttgcatctga agcaaagact gtacatttca 4860 atgcaccaca atttttaaac cgcaaggtct ttttattttc ttctacaact tcgaagttgc 4920 ctcgtctgtg acaatatgtg gtcttgttga agtaaaatag tgcacattac tgttaatgaa 4980 cactgtagga ggctagattg tgaaggagga atgatttagt ttattccgag cttctagcct 5040 cttggtgtga ggtgttagaa gaattggcac acgcaggcag tgaaatgatt caggacacag 5100 ccttcctttg atgcttccca aactgaaagg agggtttgtt ggtgggggat attaagcatc 5160 aggagaaaat tccagtaggt ttttcattag tccatatggg ggaaaatata aaatgaggag 5220 agggtaagga gaaaaaaata gtgttgtcag agaggacatt ataaaggaaa aacgcaaaac 5280 tttcaagaca ctaaactttt gtacaattgg aagtaatgca ataaaataaa ataagagcat 5340 atgcaacatt ttttaatggt gaggtgccac atatacaaaa aatacaacag gtttattgtt 5400 tctgttgtca ttttaagcat aataggcatg acatacttag aatttaagtt ttctatctcc 5460 tggaggtcac tcccagacag taattttatg actagtacct ttctctacca ctcccccacc 5520 agcattgttt ttggacagat taggcctttg tttgacatat atgtatgctg atacattcag 5580 ttttctttat tataacattt tccatgcatg gcaaacatta ttgaattcca tcattaggct 5640 agtggaattt atgaattctt agtcttgtgc attttccaga atttggaaat ccgtaggttt 5700 atttgtgaga cagcctacca tacacccgtt ctcagattaa tgattccgtg taaattatag 5760 tttagacatt gttctctgga gaaaacgtag gaccagataa aacattgaaa atctacagct 5820 ccttggggga aagcaaagat gctgattagg aatgctatag ggctgaggca tatatctcag 5880 ccctttatat tgtgcatgca caccaattag tttttcagta attcacagta cttaaggaaa 5940 gaatacccca agtcaggttt catcttttat tcatggtgct tctaatttcc atggttgatg 6000 ttaaagtgga taagataaaa ggaggaaaat gaaccacgta caaattagct ccgtaagtat 6060 tggctgcaaa tgaaggaaag caaaggatga aaggtctcca aattaaatta aagacgcaac 6120 aggcaatctg tgttcaatta ttcctccctg gtaactggaa atggtgcttt gtcatctcat 6180 acagctaact aattttcagc ctttcctttc aactgatctg cattgccagg aaagaaatat 6240 ttgcattgaa tactcttatg ttaacagcca aatccatttg caaatgaagt ttagtgtgaa 6300 tcttagctac agaaatgggc ctgttcattg gagaaggagg tggtggggtt tttcaggttt 6360 caaaaatctc aacagctact cctcttcttc ctgtggcatt gatggggttt tgcactgtgg 6420 atcacaggta atcctgcctc cctgtgcatc ctatccatct ccctctctgg aaaagcttac 6480 ttgcaaagga tttaagctgc tttaataaaa gccacaacta cagataaatg tgactttatt 6540 tttagtcatg ttttcaataa cactattgat cagatttctg tttttcacca gaaaaccttc 6600 aaagctttct taaatttaaa acagatgctt ttcaggtgaa gttggtttca cctctggaaa 6660 tcaattgaat tgaaatacta aaaaaaaata gcactttcca agcaagaaaa aaaaaaactt 6720 tagaaatttt gaagttaatg aaataagact ggaagacagg ctggctctca gacacttaag 6780 gggttatcaa aggagtcagt gactgagatt gcatctagaa gatgtttctg gaaagcaatg 6840 aactaaagaa gccccttgct tgaatctttt gttgggtgct attcagctat ttgtgtacta 6900 ggcagataaa ggagttttga ggattaattc tgtcttgctt ttttttcaga agtatttgtg 6960 tgctttccct ctttacctgc tgcttattgg agtcataatc tgaaggatat acgagtttac 7020 aatgtttggc cctttccccc ctaaatagta cttctattta tgtttcctga aaggaaacat 7080 gagatttacc ttttaccctt ggtacattta ctttatttag ggtaattctt tttaaagtga 7140 aggaacctag aagattttct tgggtggaac attaaaagac cattttacct gaaatgtgcc 7200 ccttgtattt tatttctttc cctctgaaag ctattaagag tcatgccata ggtctgataa 7260 aatgtgagaa ttttatagaa atgtatatat gaaactacag ccaattttct taggctataa 7320 atgttaggtt ttctttattc tccctgggtg tacagtattt gaattatggt tcgtttttga 7380 taagagtttt ctttggagac cagtagtttt catttggatg ttgatatttt aatggaccag 7440 aaataacaat ttgggcctaa agatgttctc ccttctgcct cttcctattg tctttttcca 7500 tcattcttcc tctctcaacc tacttttcac tgatgattct gtctcgtgaa tagacatgaa 7560 aacatgttcg gattattgct tggtgcaatg aatgttgtat cttttaaaca tttaggctgt 7620 tgctgcttct tggaaaccat tgtgggtggc tccctttttt ctgtctactc tgcttgcctc 7680 taaattacaa gctggtaaat tttctcttgt gtgaacagta atggaatagt aattgctttt 7740 aaggcatgct gcaaaagcaa aatagaaaat ccaaagtcaa tggtcttatt tattaactat 7800 ttcaggtggt attcaagtag tggtaggttt tttgttgtcg tcattgtttt ttgcacagaa 7860 ttcaatattg tacagatatg tctatttaaa tgttttggac tctggaatat tcagcctgtg 7920 taagttaaca catatatatt tgattcacag tgcttgtgaa gcatgaaatt taagttttgc 7980 atggagctca aattctccca ccaaaataca aataaaattg ttataccagt acatagatag 8040 atgcataggt aggtttagaa tacatctcca aagcacagaa ataaaactaa gataatttgt 8100 tgttcatgcc tggtaatttt tgcatacaat tttaaataaa tgttaatacc ctatatcatg 8160 gatacaagca aaatatagtt gacataaatt gagcatttac tataggcaaa gaagtaccct 8220 tagggtttta cgtgtattat ttaatcttca taacaccctg aggtaggtaa tagtactgtc 8280 tctgtttgtt tgctgtttta atactaatgc taatatttag ttatggacgc agtcccttgg 8340 tgagtacaga ggacttaaac aaatgcaacc tgacggtaat atagttgttt acctctgatc 8400 tatataatca gtatccccgt ttttaacaga agcactagga agtaacttga aaatgtcatg 8460 tagctgttat gtggcagaga tagaattttg aacccaggtg ccctggttct agcgcctgca 8520 ttgttgactg tcctgaaact catgtcattt aacacatgaa aatatgtcat cattataata 8580 agtaaagcac ctgattagca ggagccttgt gatattatta ctgtcttctg ccatttttga 8640 attcaggtct ttcttcttgg ttatgtcaac tcttagcttt ctcctttgac cttttgttac 8700 ttttttcctt tttgttgtta atgtattaca ttgaaaaata aaattttttt tttttgcaag 8760 aaaatgtact ttattagctt tctttgttct gtgggttgat cttcctggat atttctttat 8820 tttattttat tttattatta ttatacttta agttttaggg tacatgtgca caatgtgcag 8880 gttagttaca tatgtataca tgtgccatgc tggtgtgctg cacccattaa ctcgtctttt 8940 agcattaggt atatctccta atgctctccc tccccccaac ccccacccca caacagtccc 9000 cagagtgtga tgttcccctt cctgtgtcca tgtgttctca ttgtnnnnnn nnnnnnnnnn 9060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnntgt 14160 gggatataat ctcctggtgc gccgtttttt aagcctgtca gaaaagtgca gtattagggt 14220 gggagtgacc tgattttcca ggtgccctct gtcacccctt tctttgacta ggaaagggaa 14280 ctccctgacc ccttgcgctt cccgagtgag gcaatgcctc gccctgcttt cgcttgggca 14340 cggtgtgctg cacccactgt cctgcgccca ctgtctggca ctccctagtg agatgaaccc 14400 ggtacctcag atggaaatgc agaaatcacc catcttctgc gtcgctcacg ctgggagctg 14460 tagaccggag ctgttcctat ttggccatcg tggctgccag ctcaaaatta tatgttaata 14520 tatttgttgt gtagactata atgttttgat atagggatac attgtggaat tactaaagct 14580 aattaatata ttcattacct tacaatttta tcattttttg tgtgtgtggt gaaaacattt 14640 aaaatctcct cagcagtttg caggtgtaca atacattgtt aataactata gtcaccaatt 14700 tgtgttgcag ttgatcttct aaatgtattc tttctaactg aaattttgta tcctttgacc 14760 aaacatttcc ccaattccta cccctacctt ttttctcctg ataatatttg gtcttgtgaa 14820 aattttatct catctctcac tatctctgat ttatagacca tttgatattt atttttacaa 14880 gctagataaa aactgccaag ttagtatata aactgtagtc ttttattttt taaactacgt 14940 attcttgtga ataagaattg ataacttcta tagatattaa aattaagttt ttcctcattt 15000 gctatgtata catcaacaac tctgtctcct aggcgtgtat catgtcaggg caggcagaac 15060 tgaacggaag aataattttc agggtagaat gggagaaaat acagaattat gtgaagcagg 15120 ctcaggggtt tattattgaa acagaggaga ggagctgggg gcctgagcca atttgcgctg 15180 aagccaatgt ggctgagatg gctccttctg ggaacagaga tgtgcttgga ggagccttgg 15240 ttctcctcac caatataccc cttatgttgg ctgctgcagg cagcactacc aggttcattt 15300 gactgctcac ttgttggtag aaggtttgtt aattttcttg tccgatttct tctgagatta 15360 ggaatttcct tggattaaaa acaaaataaa tcattggttg agtctttttg caggttaaaa 15420 aaaacaagaa aacttggagg tggatttgct gaggaggaca gccatgttct ctaccagaag 15480 tcagattggg tctttttcct aaaacaacta aaaacgcagt agtttgttta ctttttaaaa 15540 tccgtctcct ccacctgtgg aaagagaact ttaaacacta tttatgttac atgctgaatc 15600 taagactgtt gttccttcag cttgaactgc agatattcct aaattgtacc tgcatctcta 15660 gtgaaagctg agtgtgctaa atgctgacca ttctccatta actgagattt cagttatcta 15720 agagatttgc atcttagcag tactactgta ttactctgta agattccttc tacctacctg 15780 agctcaaact tcatcctatt cagaagatgt tttaatataa caagtcagta ggccttctct 15840 accacttaac aaatattaat tgagcaccta ctgtgtacaa ggcactgtgc ttaattaagt 15900 cagggagtat ttcagctccc tgaaacagag agcatctcag ccctcccaga ttgattctct 15960 ggactaaata tgcatggtag cttcagtgcc tgttgagaga aatgaatgtg tctgggagtg 16020 taaagcagag gtccctaatg gagttgttga ttgtaggagt aattgtggag gaagtgacat 16080 ttgagctgag aagtaaagaa caacagggcc tagtgggtga tgggacaggg atgaaggagc 16140 cctccagaga gagaaaacca tatttgtgga cctgaatact cagagacctg aaggatgttc 16200 ttgatggcaa gaggctgggt ggtgagaaga tgaggtggag aaataggcag ccacttttcc 16260 atcttctgca tgtcaaagtg ctgttctcat ctttgagaga agctgtcctg taaagaactc 16320 ttcaggttct gtgtatttgg aagtggcatc taaatcatgc ccattggaat cttcttcctg 16380 gatactatca tgtctttatg taaacttgtg aatttaaaag tatacatagc agtgtcggag 16440 caggctttcc tttttatttt tttgcatctg gttttcagaa tgcccattag gcaaattttt 16500 gacctttaac tttttcaata ggccttggat aaggtgggtg gtaaattgat aggaaaacta 16560 tagataagta tttgtccatc tagttgtacc aaagaagtgg taatatagac ttttgtggat 16620 cctttagagc acctgatatc acattaaata atatgatacc tgaatatgta tttcagttgt 16680 ttctcccact agaataccag ggtaggaatt ttcttttgtt tactgttgta tctgtagtgt 16740 ccagagcagt gcctagcatg cagtgaatgc ttattaaata ttttttgaat gaatgaatta 16800 taagacactt ggaagctgag ggaatttatt ataaacagag tttaatccct gaaaggagtc 16860 ctgcacagag attgtcaatc aaatcatagt tttgaagtct gtgttgtatg tctaagattg 16920 tattgagccc tttaaataga aactggaaga taaacgtggt ccctactctg attctaagag 16980 cttttatact aaaaggaaag agaatgtcat gagcatttat gtatatagca aggcattacc 17040 atcaacagcc attaaaaggg gaggtttgtc aaggtggtcg tgagtcagtt gagtatttgg 17100 cctcttcaca cgtgtgagag gctggaggct ggtggggagc tcacataggc gtaacagccc 17160 atgttcaaat ccagcttcac tgcttagtgt ttgcattaca ttggcaaggg ttgactgcct 17220 cgagtgattg ttttaagtgc tctaggcaag taataaatat tagttctttt tgccttgttc 17280 tttccactat gggtgactgc cctaattgag ggtataccag ataattgcac tgtcttgatt 17340 tgttaagtgc ctctaaatgt tcttctcctg acatctggac tatgtttcta gaggcctagg 17400 gggaggagga ggtaaaggca cttgactttc ccaaattagt ctgtacccaa gtggaactgc 17460 ataaattggg taccttaatt acaccattgg gtattactag ggtgattaca gggacaagag 17520 ttaaataggg cttgtctaca aagagtcctg ggaggaagtg agatgtgaga gaactaaaag 17580 catctaaagg acattttaga tggagaggag gtgaagggtg ttctcctgtg ggttctgact 17640 tctgatgcta actcctaacg gtgaaagcta ggaagttgac ttacatgtag ctcagaggag 17700 gatgctaacc tggctcactg caacttccac ctcctggatt gaagcgattc ccctgcttca 17760 gcctcctgag tagctaggat tacaggtgca agccatcatg cctggctaat ttttgtattt 17820 ttagtaaagg tggggtttca acatgttggc caggttggtc tcaaactcct gacctcaggt 17880 gatgcaccca cctcggtttc ccaaagtgct gggattacag gtgtgagcca cggtgcctgg 17940 ccctaggtgt ggtttttaat tactatgtaa tcccagggcc cagtgtggac ctaggacata 18000 gttactacat gatacagtga ttatcgttgg cacaaatgaa tgaatcaaag agatagtatg 18060 ttacaaagga gagggcacga aatagatttt ttttttgagc acatgattag aagcagtatt 18120 ttaaggtcat ttcctgggct gggaaggtta ggaatattct tctgaccagc acctgttgga 18180 ggaggtggaa aggatcccag atgccagcct tctctgtgtc cacccttctc ccagccttgc 18240 ctggggacac tgagcccctt gctgggcttc aagtgcctga tgcttttttg ggaagcccca 18300 taagcttgct tctctttttg caaacattgc ttaagttttc attaataaaa acattctaaa 18360 gcttatttag tccagaatga ggaaaatgta ggagagagag agggtgtggg tgtgtgtgtg 18420 gggcgagggg gtgtctgtgt gtggttttac ctacctatgt ggttgcccct tcttacctgt 18480 gaatgcagtg gctgctttcc ttgttccttc tccacagacc ctgtaccctc catttcagtt 18540 tctggcaaga attgtggcca gacccaggga tgctgatgca gtggtcccca acattttctg 18600 gtcacataat ttctaaaaga attttgtaac attaaccctc tgggatattt tttaattgac 18660 atctacattt tatttttttt attattttat tattattata cttaagtttt agggtacatg 18720 tgcacaatgt gcaggttagt tacatatgta tacatgtgcc atgctggtgt gctgcaccca 18780 ttaactagtc ttttagcatt aggtatatct cctaatgcta tccctccccc caacccccac 18840 cccacaacag tccccagagt gtgatgttcc ccttcctgtg tctgtgttct cattgttcaa 18900 ttcccatcta tgagtgagaa catgcggtgt tggttttttg tccttgcaat agtttactga 18960 gaatgatgat ttccaatttc atccatgtcc ctgcaaagga catgaactca tcatttttta 19020 tggctgcata gtattccatg gtgtatatat gccacatttt cttaatccag tctatcattg 19080 ttggacattt gggttggttc caagtctttg ctattgtgaa tagtgctgca ataaacatac 19140 atgtgcatgt gtctttatag cagcatgatt tatagtcctt tgggtatata cccagtaatg 19200 ggatggctgg atcaaatggt atttctagtt ctagatccct gaggaatcgc cgcactgact 19260 tccacaatgg ttttactagt ttacagtccc accaacagtg taaaagtgtt cctatttctc 19320 cacatcctct ccagcacctg ttgtttcctg actttttaat gatcaccatt ctaactggtg 19380 tgagatggta tctcattgtg gttttgattt gcatttctct gatggccagt gatgagcgtt 19440 ttttcatatg tcttttggct gcataaatgt cttcttttga gaagtgtcta ttcatgtcct 19500 tcgcccactt gttgatgggg ttggttggtt ttttcttgta aatttgtttg agttcattgt 19560 agattctgga tattagccct ttgtcagatg agtaggttgc gaatattttc tcccattttg 19620 taggttgcct gttcactctg atggtagttt cttttgctgt gcagaagctc tttagtttaa 19680 ttagatccca tttgtcaatt ttggcttttg ttgccaatgc ttttggtgtt ttagacatga 19740 agtccttgcc catgcctatg tcctgaatgg taatgcctag gttttcttct agggttttta 19800 tggttttagg tctaacgttt aagtttccat cttgaattaa tttttgtata aggtgtaagg 19860 aagggatcca gtttcagctt tctacatatg gctagccagt tttcccagca ctatttatta 19920 aatagggaat cctttcccca ttgcttgttt ttctcaggtt tgtcaaagat cagatagttg 19980 tagatatgca gcgttatttc tgagggctct gttctgttcc attgatctat atctctgttt 20040 tggtaccagt accatgctgt tttggttact gtagccttgt agtatagttt gaagtcaggt 20100 agtgtgatgc ctccagcttt gttcttttgg cttaggattg acttgacgat gtgggctctt 20160 ttatggttcc atataaactt taaagtagtt ttttccaatt ctgtgaagaa agtcattggt 20220 agcttgatgg ggatggcatt gaatctataa attaccttgg gcagtatggc cattttcaca 20280 atattgattc ttcctaccca tgagcatgga atgttcttcc atttgtttgt atcctctttt 20340 atttcattga gcagtggttt gtagttctcc ttgaagaggt ccttcacgtc ccttgtaagt 20400 tggattccta agtattttat tatctttgaa gcaattgtgc atgggagttc actcatgatt 20460 tggctctctg tttgtctgtt attggtgtat aagaatgctt gtgatttttg tacattgatt 20520 ttgtatcctg agactttgct gaagttgctt atcagcttaa ggagattttt ggctgagaca 20580 atggggtttt gtagatatac aatcatgtcg tctgcaaaca gggacaattt gactccctct 20640 tttcctaatt gaataccctt tatgtccttc ttctgcctaa ttgccctggc cagaacttcc 20700 aacactatgt tgaataggag tggtgagaga gggcatccct gtcttgtgcc cgttttcaaa 20760 gggaatgcct ccagtttttg cccattcagt atgatattgg ctgtgggctt gtcatagata 20820 gctcttatta ttttgagata cgtcccatca atacctaatt tattgagagt ttttagcatg 20880 aagcgttgtt gaattttgtc aaaggccttt tctgcatcta ttgagataat catgtggttt 20940 ttgtctttgg ttctgtttat atgctggatt acatttattg atttgcatat attgaaccag 21000 ccttgcatcc cagggatgaa gcccacttga tcatggtgaa taagcttttt gatgtgctgc 21060 tggattcggt ttgccagtat tttattgagg atttttgcat ccatgttcat caaggatatt 21120 ggtctaaaat tctctttttt ggctgtgtct ctgcccggct ttggtatcag gatgatgctg 21180 gcctcataaa atgagttagg gaggattccc tctttttcta ttgattggaa tagtttcaga 21240 aggaatggta ccagttcctc cttgtacctc tggtagaatt cggctgtgaa tccatctggt 21300 cctggactct ttttggttgg taagctattg attattgcca caatttcaga gcctgttatt 21360 ggtctattca gagattcaac ttcttcctgg tttagtcttg ggagggtgta tgtgccgagg 21420 aatttatcca tttcttctag attttccagt ttatttgcgt agaggtgttt atagtattct 21480 ctgatggtag tttctatttc tgtgggatcg gtggtgatat cccctttatc atttttttgt 21540 gtctatttga tttttctctc ttttcttctt tattagtctt gctagtggtc tatcaatttt 21600 gttgatcctt tcaaataacc agctcctgga ttcattaatt ttttgaaggg ttttttgtgt 21660 ctctatttcc ttcagttctg ctctgatttt agttatttct tgccttctgc tagcttttga 21720 atgtgtttgc tcttgcttct ttagttcttt taattgtgat gttagggtgt caattttgga 21780 tctttcctgc tttctcttgt gggcatttag tgctataaat ttccctctac acactgcttt 21840 gaatgtatcc cagagattct ggtatgttgt gtctttgttc tcgttggttt caaagaacat 21900 ctttatttct gccttcattt cgttatgtac ccagtagtca ttcaggagca ggttgttcag 21960 tttccatgta gctgagcggt tttgagtgag tttcttaatc ttgagttcta gtttgattgc 22020 actgtggtct gagagacagt ttgttataat ttctgttctt ttacatttgc tgaggagagc 22080 tttacttcca actatgtggt caattttgga ataggtgtgg tgtggtgctg aaaaaaatgt 22140 attttctgtt gatttggggt ggagagttct ttagatgtgt attaggtccg cttggtgcag 22200 agctgagttc aattcctgtg tatccttgtt aactttctgt ctcgttgatc tgtctaatgt 22260 tcacagtggg gtgttaaagt ctcccattat tattgtttgg gagtctaagt ctctttgtag 22320 gtcactcagg acttgcttta tgaatctggg tgctcctgta ttgggtgcat ttatatttag 22380 gatagttagc tcttcttgtt gaattgatcc ctttaccgtt atgtaacggc cttctttgtc 22440 tcttttgatc tttgttggtt taaagtctgt tttatcagag actaggattg caacccctgc 22500 ctttttttgt tttccatttg cttggtagat cttcctccat ccttttattt tgagcctatg 22560 tgtgtctctg cacgtgtagt atgggtttcc tgaatacagc acactgatgg gtcttgactc 22620 tttatccaat ttgccagtct gtgtctttta attggagcat ttagtccatt tacatttaaa 22680 gttaatattg ttatgtgtga atttgatcct gtcattatga tgttagctgg ttattttgct 22740 cgttagttga tgcagtttct tcctagcctt gatggtcttt acgttttggc atgattttgc 22800 agtggctggt accggttgtg cctttccatg tttagtgctt ccttcaggag ctcttgtaag 22860 gcaggcctgg tggtgacaaa ttctctcagc atttgcttgt ctgtagagta ttttatttct 22920 ccttcactta cattttaaaa atcattttaa actgttgcaa aacttttgat ttccagtgca 22980 ttataaatgt tgacatttgt aatgaaattt aaatatatcc agtagatcta taaatattgt 23040 taacagtttg atacccacca ccattcattt aataaacata cttttcaatg acatttggat 23100 ggagatatat atatacacat atatatattt tttaagtgag acaggggtct ctctctgttg 23160 cccaaactgg agtgtagtgg tgcgatcaca gcttactgca gcctccacct ccctgggtca 23220 agcaattctc ctgcctcacc ctcctgagta gctgggacta caggcacatg ccaccatacc 23280 tggctaattt ttatttttat ttttttttgg tagagacagg gtcctcctat gtttccctgg 23340 ctggtctcaa gctcctggct tcaagcagtc ctaccgcctc atctttccaa agtactggta 23400 ttactggtgt gagccaccac acctggccac gtttggatat tttatattgc tctttttttt 23460 ttctatcttc attcctcctc agaattttat cttagcataa tattttgagg ttcaaagtca 23520 tctccctatt acacatatct acaatgacaa aaagaatata gatagaattt aaagttttat 23580 aatcctgtgg ctatcagcct ctgtcttaga aaagcttcag gactgactta ccattacaat 23640 tagtattggt aaacaattaa tcaaaacaac aataaatgtt acagggttgg gtaattaact 23700 attaaacata aaagctacat ttattagatt tcttagtgac atggatggag cattttgttg 23760 ttgattatat attctgtatt gacaaatatt acagattgct agagatagaa tttagtataa 23820 atttcattcc tattaggtca actttttttt acgtaaagaa gtaattaagg tgggaatcat 23880 tttcttacag cagtgtttct cagttttttg aactctcaag ctttaagcca aaaattacat 23940 ggtgatctga cagcaaaaac tattttaatg atttattaat gatattaggt cacctccagt 24000 tttgttgaat gcaaatagtt agaaacctct ggttgcccca aaggatttat tacccattca 24060 ttagagacat tggctttctt aataaagata gcaatatttc tgattacctt tgtttggtgc 24120 cctggaggtt atattaggtg ggacagtgca tttaataaca atacaggatg ggtgagcagt 24180 caggcccttt gtgaagtggg agaagggtga tgtgaagtgg ggtggggcgg gggtgggggg 24240 cagagcgaga gagaagcatg gagaacctgc ccctatccaa gtggatcagt tttaggaaag 24300 cttatgccgt atgtagggat gccaagggtc tgggaattta ttccctaact taaagttatc 24360 tgcattcaca tattccatgg agagttgtat gcattcaggt atcatgcact cccagtctga 24420 aaaactgctc ttttggttga aagagtataa ggacttggaa gatggggcct ctaggcctga 24480 ctgagccaaa ggctcccatg acctcaaggg aactgactag tgcttttttc attttttact 24540 gcgttctggg tcccaccctt ggcattcaga tgtgtgcatg agctgctgtt taattttcat 24600 catctgcttt acaaatgagg aaaagccctg atatccagct ccctcatagg acctgataat 24660 aatacatatg gttctgagaa ttaaacgagg taataggtac gtaaagtggg taaacagagt 24720 aacttcactc tctatttgta cgtctctcag aagtctgggg caacaggagc tgtggaaggc 24780 tccagaagac aagatgtcac aaagccacct agctactgtg gctgtaccaa tcctgacagt 24840 catagcggcc ccaaggtgct gctctgttgg ctgatgggaa tgcagattta cgtatggtcc 24900 atatatggac gtgttgtttt acatctaaat aaacttggca ttgcaagatg aaaatgccag 24960 tatcattttt aaaggtcaag tgacagtttg ggaagatttt tataactttg ataagttcct 25020 cagctacatc tgttcacact ggcaagagtg ctagagacat tcttgggaag tcttttctat 25080 cagaggagct gcagccttgc tctgcagaaa agttctgatg acagtcatca gagtatttgg 25140 ggctcatgca ctaactgtta attattttca tttttcatta atgaaaattt attttaattt 25200 ttttcattct tctttaatga atttaactat ttgcctctac ctttgcttcc tgctttgtat 25260 actgataaga caggttttgg ggtttttatg ttttctggtc ttgcttttcc cctttgtttt 25320 aggatttaaa aaacaaaaag gttaagaggg taggttgaca gtgcttgcag tttctgtttt 25380 acagcaatcc cagctgttcc tctgaaaatt ggttgattgt cctatagttt aaaatatata 25440 gatgttcatt tgattggctc ttgtaaatct agtgacattt aaaagtacat ggcagctagt 25500 catggtggct cacacttata atcccagcac tttgggaggc tcaggcagga ggatggctta 25560 gcccaggagt tcgagaccag cttgggtaac atcatgagac cccaactcta caaaaatttt 25620 taaatagttg tgagtggtgg catgtgcctg taccctcagc tacttgagag gctgaggtgg 25680 gaggattgtt tgagacctgg agtttgaggt tgtggtgctc tatgattgca ccactacact 25740 ccagcctgga tgatagactg agaccctgtc tcaaaaaaga aaaagaaaaa agttcatgtc 25800 tattctgttt tttgttatgt tactttcctc taaacatgaa gaaaagcagt gaggagaaac 25860 aaaaattgag tattcctgtg ttttttatca tagtaaaata tacatttgcc attttatatt 25920 tatcctgata accttcagtg ttgtaaaata cacattcaca attattcatt taacctgata 25980 acctctaatc tcttttccat gaatcggact aggtacctca tataagtgga atcataccat 26040 atttatactt ttgtgtctgg tttatttcac ttaatgtcct taaggttcat ccacattgta 26100 gcatgtctca gaatttaatt cctttttggg gctgaataac attctattat atgtatattc 26160 cacattttgc ttatccattc atctcttgat aggaagttgt gttgctttca cattttacct 26220 attgtgaata atactggtac caatattagt gtacacataa tctgagtccc tgctttccgt 26280 tcttttgggt atatacacag aagtaaaatt gctagacaca atggtacttc tatgtcagtt 26340 ttttgaagaa ctaccctact gttttccata gcagttacac cattttacat ttctatcacc 26400 agaccacgag ggtaacaatt tctccacacc ttagccacca cttgttattt tttgtttttt 26460 gggtaatagc catgcaaaaa aatatgaagt aaagtgaaat atcattatga aaggtgtgaa 26520 gtgatatgtc tttgtgattt tgatttgcat ctctcctata attagtgatg ttgaacatct 26580 tttcatgtgc ttattggcca tttgtctgtc ttctttgggg aaatatctct tcaagcctat 26640 tctgcccatt tttgaattgg gttttgttgt ttttaggaat tctttagata ttttggatat 26700 tagtcactta tcagatatgt gatttgcaaa tgaggatggc ctttttattt tgttgattgt 26760 gttctttgca caatagtttt tatttttgat gaaatataat tttctatttt gtgcttttgg 26820 tgttatatat ctaagaaatc atgccaaatt caatgtcatg aaggctttct cctgttttct 26880 tctaatagtg tgtagtttta gctcttttgt acgtggtgtt gggtaagggt tcaacttcat 26940 ccttttgcat gggaatatcc agtttttttc ccagctgcat ttgtcaaaaa gactcttctt 27000 tcttccgttg aatgatctca gtggaagtca ctgatctcat caaaaatcac tgaccatatg 27060 tgggagcata accacctgat gggttcttct tgcctgctgc ccaaatatag ctggtttatc 27120 aagatgggaa ttgcaataga gaaagagctt tacacatgta cagctggcta aatgggagac 27180 tggagcttta ttattaaata aataagcctc ccccaaaatt tggatgctag ggttttctct 27240 ttttttttct ttttttatta ttatacttta agttctaggg tacatgtgca caacgtgcag 27300 gtttgttaca tatgtatata tgtgccacgt tggtgtgctg cacccattaa ctcatcattt 27360 acattaggta ttctcctaat gctatccctt ccccctcccc ccaacccact acaggcccca 27420 gtgtgtgatg ttccccacca tgtgtccaag tgttctcatt gttcagttcc cacctatgag 27480 tgagaacatg tggtgtttgg ttttctgtgc ttgagatagt ttgctcagaa tgatggtttc 27540 cagcttcatc catgtcccta caaaggacat gaactcactc ttttttatgg ctgcatagta 27600 ttccatggtg catatgtgcc acattttctt aatccaatct gtcattgatg gacatttggg 27660 ttggttccaa gtctttgcta ttgtgaatag tgccgcaata aacatacgtg tgcatgtgtc 27720 tttataatag catgatttat aatcctttgg gtatataacc agtaatggga tggctgggtc 27780 aaatggtatt tctagttcta gatccttgag gaatcaccac actgtcttcc acatggttga 27840 acgagtttac actcccacca acagtgtaaa agtgttccta tttctcccat cctctccagc 27900 acctgttgtt tcctgacttt ttaatgatcg ccattctaac tggtgtgaga tggtatctca 27960 ttgtggtttt gatttgtatt tctctgatgg ccggtgatga tgagcatttt ttcgtgtgtc 28020 tgttggctgc atgtcttttg agaagtgtct gttcatatcc ttcgcccact ttttgatggg 28080 gttgtttgat tttttcttgt aaatttgttt aagttctttg tagattctgg atattagccc 28140 tttgtcagat gggtagattg caaaaatatt ctcccattct gtgggttgcc tattcactct 28200 gatggtagtt tcttttgctg tgcagaagct ctttagttta attagatgcc atttgtcaat 28260 tttgactttt gttgccattg cttttggtgt tttagtcatg aagtccttgc ctatgcctat 28320 gacctgaatg gtatagccta cgttttcttc tagggttctt atggttttag gtcttacatt 28380 taagtcttta atccatcttg aattaatttt tgtataaggt gtaaggaagg gatccagttt 28440 catctttcta catatggtta gccagttttc ccagcaccat ttattaagta gggaatcttt 28500 tccccatttc ttgtttttgt caggtttgtc aaagatcaga tggttgtaga tgtgtggtat 28560 tatttctgag ggctctgttc tgtgccattg gtctatatct ctgttttagt atcagtacca 28620 tgctatttgg ttactgtagc cttataatat aatttgaagt caggtagcaa gatgcctcca 28680 gctttgatct ttttgcttag gattgtcttg gcaatgtggg ctcttttttg gttccatatg 28740 aactttaaag tagttttttc caattatgtg aagaaagtca ttggtagctc aatggggatg 28800 gcagttaatc tataaattac cttgggcagt atggccattt tcacaatact gattcttcct 28860 atccataagc atggaatgtt cttccatttg tttgtcctct tttatttcat tgagcagtgg 28920 tttgtagttc tccttgaaga ggtccttcac atcccttgta agttggattc ctgggtgttt 28980 tattctcttt gaagcaattg tgaatgggag ttcactcatg atttgtctct ctgtctgttt 29040 ttggtgtata ggaaagcttg tgattttgca cattgatttt gtatcctgag actttgctga 29100 agttgcttat cagcttaagg agattttggg ctgagacgat ggggttttct aaatatacag 29160 tcatgtcatc tgcaaacaga gacaatttga cttcctgttt tcgtgattga atagctttta 29220 tttctttttt taaatatata tttttattat actttaagtt ctagggtaca tgtgcacaat 29280 gtgcaggttt ttacatatgt atacatgtgc catgttggtg tgctgcaccc attaactcgt 29340 catttacatt aggtatatct cctaatgcta tccctccccc ctctccctac cctggtgtgt 29400 gaagttcccc ttcctgtgtc caagtgttct cattgttcaa ttcccaccta tgagtgagaa 29460 catgcggtgt ttggtttttt gtccttgcga tagtttgctg agaatgatgg tttccagctt 29520 catccatgtc cctacaaagg acatgaactc atcctttttt atggctgcat agtattccat 29580 ggtgtatatg tgccatgttt tcttaatcca gtctatcatt gttggacatt tggtttggtt 29640 ccaagtcttt gctattgtga atagtgcctc aataaacata catgtgcatg tgtctttata 29700 acagcatgat ttataatcct ttgggtatat acccagtaat gggatggctg ggtcaaatgg 29760 tatttctagt tctagatccc tgaggaatca ccacactgtt ttatttcttt ctcctgcctg 29820 ccagctcctc ctggtacctc tgatagaatt cagctgtgaa tatgtctggt ctgtggcttt 29880 tcttgcttgg tacactatta attattgcct caatttcaga gcctgttatt ggtctattca 29940 gggattcaac ttcttcctgg tttagtcttg ggaggttgta tgtgtccagg aatttttcta 30000 tttcttctag attttctaat ttatttgtgt agaagtgttt atagtattct ctgatggtag 30060 tttgtatttc cgtgggattg gtggtgatac cccctttatc attcttatta catctatttg 30120 attcttctct cttttcttcc ttattagtct gctagtggtc tatcaatttt gttgatcttt 30180 tcaaaaaacc aattcctaga ttcattgatt ttttgaagtt tttttttttg tgtctctgtc 30240 gccttcagtt ctggtctgat cttagttatt tcttgcgttc tgctagcttt tgaatgtgtt 30300 tgctctggct tctccagttc ttttaattgt gatgttaggg tgtcgatttt cagtctttcc 30360 tgctttctct tgtgggcatt tagtgctata aatttccctc tacacactgc tttgaatgtg 30420 tcccagagat tctggtatgt tgtgtctttg ttctcgttgg tttcaaagaa catctttatt 30480 tctgccttca tttcattatg tacccagtag tcattcagga gcaggttgtt cagtttccat 30540 gtagttgtgc agttttgaat gagtttctta attctgagtt ccaatttgat tgcactatgg 30600 tctgagagag tttgttgtga tttctgttct tttacatttg ctgaggagtg ccttacttcc 30660 aactaagtgg ttaattttgg agtaagtgca atgtggtgct gagaagaaag tatattctgt 30720 taattttggg tggagagttc tgtatgtgtc tattaagtcc acttggtgca gagctgagtt 30780 caagtcctgg atatccttgt taaccttctg tctcattgat ctaatattga cagtggagtg 30840 ttaaggtctc ccattattgt ctcccattat tattgtttgg gagtctaagt ctctttgtag 30900 gtttctaagg actcgcttta tggatctggg tgctcctgta ttgggtgcat atatatttag 30960 gatagttagc tcttgttgtt gaattgatcc ctttaccatt atgttaacgg ccttctttgt 31020 ctcttttgat ctttgttggt ttaaagtctg ttttagcaga gactaggata gcaacccccg 31080 gtttttttgc tttccatttg cttggtagat cttcctccat ccctttattt tgagcctatg 31140 tgtgtctctg catgtgagat gggtctcctg aatacagcac actgatgggt cttgactctt 31200 tatccaattt gccagtctgt gtcttttaat tggggctttt agcccattta cattgaaggt 31260 taatattgtt atgtgtgaat ttgatcctgt catcatgatg ttagctggtt attttgctca 31320 ttagttgatg cagtttcttc atagcatcga tggtctttac aatttggcct atttttgcag 31380 tggctggtac tggttgttcc tttccatgtt tagttcttcc ttcaggagct cttgtaaggc 31440 aggcctggtg gtgacaaaat ctctcagcat ttgcttgtct gtaaaggatt ttatttctcc 31500 ttcacttatg aagcttagtt tggctggata tgaaattctg ggttaaaaat tcttttcttt 31560 aagcatgttg aatatcggcc ccctctctct tctggcttgt agtgtttctg ctgagagatc 31620 tgctgttagt ctgatgggct tccctttgtg ggtaacccga cctttctctc tggctgccct 31680 taccattttt tccttcattt caaccttggt gaatctgaca attatgtgtc ttgggattcc 31740 ccttctggag gagtatcttt gtggcgttct gtgtatttcc tgaatttgaa tgttggcctg 31800 ccttgctagg ttggggaagt tctcctgcat aatatcctga agagtgtttt ccaacttggt 31860 tccattctcc ccgtcacttt caggtacacc aatgaaacgt agatttggtc ttttcacata 31920 gtcccatatt tcttggagac tttgttcatt tctttttact cttttgtctc taaacttctc 31980 ttctcacttc atttcattca tttgatcttc aatctctgat accctttctt ccagttgatc 32040 gagtcagcta ctgaagcttg tgcattcgtc acgtatttct catgccatgg ttttcagctc 32100 catcaggtca tttaagatct tctctatgct ggttattgga attagccatt tgtctaatcg 32160 tttttccagg tttttagctt ctttgagatg ggttcgaaca ccctccttta gctcggagaa 32220 gtttggtatt accaatcttc tgaagcctac ttctgtcaac tcattaaagt cattctccat 32280 ccagctttct tcctttgctg gcgaggagct gcgatccttt ggagaagagt cgctctgatt 32340 tttaaaattt ttggcttttc tactctgttt tctccgcatc tttgtggttt tatcaaactt 32400 tggtctttga tgatggtgac ctacagatgg ggttttggtg tggatgtcct ttttgttgat 32460 gttgatgcta gttttgttga tgctttgtta gttttccttc taacattcag gaccctcagc 32520 tgcaggtctt ttggagtttg ctggaggtct actccagacg ctgtttgcct gggtgtcacc 32580 agcagaggct gcagaacaac aaatattgca gaacggcaaa tgttgctgcc tgatccttcc 32640 tctggaacct tcatctcaga ggggcacctg gctgtatgac gtgtcagtcg gcccctactg 32700 ggaggtgtct cccactgggc tactcagggg tcagggaccc acttgaggag gcagtctgtc 32760 cattcccaga tctcagactc tgtgctggga ggaccactgc tctcttcaaa gctgtcagac 32820 agggacgttt aagtctgcag gtttctgctg ccttttgtta agttatgccc tgcccccaga 32880 agtggagtct acagaggcag gcaggcctgg ttgagctctg gtgggctcca cccagtttga 32940 gctttctggc cgcttcgttt acctagtcaa gcctcagcaa tggcagacgc ccctccccca 33000 gtcttggggc cgtcttgcag tttgatctcg gactgctgtg ctagcagtaa gcaaggctcc 33060 ataggtgtgg gaaccgccga gccaggcacg ggttataatc tcctggtgtg ccgtttgcta 33120 agaccattgg aaaagcgcag tattagggcg ggagtgtccc gagtttccag gtaccatctg 33180 tcacggcttc ccttggctag gaaagggaat tccctgaccc cttgtgcttc cagggtgagg 33240 tgatgccccg ccctactttg gctcacactc catggcctgc acccactgtc caacaagtcc 33300 cagtaagatg aacccagtac cttagttgga aatgcagaaa tctcctgtct tctgcgtcac 33360 tcacgcagat gcatgagctg tagactagag ctgttcctct tcggccatct tgtaacgatc 33420 ctgctagggt tttttcaagg atagtttggc agggagagag atgacaaagg aatgagtgct 33480 gctgattggt tggcggtgca atcacagagg tgtgggaaat gatccttatg ctgagtccac 33540 ttctgggcga aggccgcagg actggttggc aggtcaggtg gtgccatctg gttgtcagaa 33600 ttgcaaaagc ctgaaaagac atctcaaaag gccaacctca ggatctataa tagtgatatt 33660 acctgcagga gtaattgagg aagttgcgaa tcttgtgatt tgcagaataa tgactggtaa 33720 ttgttcacat ctatgtctta gtagaattga ggctcctctc attctcccat tctggtggtc 33780 tttcattagt tttacgaaag tagtttagtt tgggggaagg gctattatca tttaaactac 33840 aaactaaatt tcttccaaag ttagcttgac ccaatcccag gaatgactaa gggcatttgg 33900 agggtaaagg caagatgcag gctggttgga tcagatctcc ttcactgtca taattttctc 33960 actgttttaa gttttgcaag ggtggttgca tagggtttat ttctgggctc tctattctag 34020 tccattggtc tatatttctg tctttatgcc agtaccacat tgctttgatt aatgtagctt 34080 tgttataagt tttgaaattg gaaagtgtga cttacttatt cttttcaaga ttcttttggc 34140 tattcagggt gccttgagat tccatgtgaa ttttaagatg tattattatt ctgttactgc 34200 aaaaaacatc attgggattt tgatagagat tgcattgact ctgtagatct tctttagtag 34260 ttttgacatt tagcaatatt aactcttcca atccataaat atggaatttt gttccattta 34320 tctttgtggt ctttaatttc tttcaccagt gtttcatagt ttttagtgta tatggttttt 34380 gcctccttgg ttaagtttat ttatgatttt ttatgctggt aggttttatt ttttaaattt 34440 tgggggaagt gttttattaa tacaaatgta tgggttacaa gtgcaatttt gttacatgta 34500 tagattgtat agtggtgatg tcaggacttt tatggtatcc atcactcaaa taacttccat 34560 tgtacccatt aagtacaccc cactcccacc tgttaagttt taataagttg gtttagtctc 34620 tgttctgtct ctgaaaaatg tcagatacta ctaacagaac taaaaaatcc aggactgaga 34680 gaaactttaa gtacatacaa caatataaac agatcttaaa aatatgatac agcatagaaa 34740 aatggaaacc aaacatattc cacaatgtca ttcagtactt ttaaattact acacagaaag 34800 tgttatacat ttcacaaaaa tgtataagaa tagcacacac aaattatcaa caaaataata 34860 cagggttttc tgaattgtct acctgtgaga tgtttctaag cagggtttta ttattgattg 34920 attgattgag acagggtctg actctgtcgc ccagattgga gtacagtggt gcaatcctgg 34980 cttgacctcc tggctcaagt aatccttcca cctcagcatc ccaagcagct gggactatag 35040 gttcacacaa ccatgcctgg ctacttcttt aaaaaaattt ttgtagagag tctcactaca 35100 ctgctcaggt ggtctcaagc tcttgggctc aagcaattct gacacctcag cctcccaaag 35160 tgcagggatt acgggtgtga gccgttgcac ctggactaag cagggtttta agtgacttgg 35220 ttcttgttta tgttgacact aaacatgcat caaagattaa ccttctaaat tctggaacac 35280 agaaagaagg gcagccctta tttcagggct gggctgatga tagaatttac ttgatgcagt 35340 ttttcataga aagatatgtg tctttttgtt ttaaacatat aagaaaagag tttccactgt 35400 ttacctggta ctaggtccca atgtctaaac caagtttgga gaatcacatc ctctgagcag 35460 cctatgttga tgatgatgct gatgacaatg atgacagaag acacttatat ggtacttatt 35520 tatgtagcag aaccatagtc ctttatatgt tttaactcat ttaatattca aaacaaccct 35580 atgacatggt acattgatgt tgttataccc atttatatgt gtggaaatgg tcatacagtg 35640 tcttattaat ttgcctgagg ccacacagct aattagtggt ggatctgagg ttggatttct 35700 aggagccaga ttcacaaatc caacactgca ctttgctttt tctctattgt ctatcgctca 35760 ttgatttttt tattctcaat tttatttcct tcctttattt ttggtttaat tttctcttct 35820 ttttttagtt tcttgagatg aaaacttaga tctttgattt ttctacctct cttcttttca 35880 atatttatta tgcttaaagc tttacacttc accctaggca ttgtttttgt tgcatcccac 35940 atattttgat gctgtatttt tatattcaga atattttcta atttatattt atttgttcat 36000 tttaaaaata attttactaa ataatataaa atgctaattt tgaaatactt aggattttct 36060 tacatttggt tttgatttct aatttaattc tatgtggttt taaaatatag tttcactcaa 36120 ttgaaattta ttgatttact ttatattcct gcatatggtt gatactggtg catgttgcat 36180 gtacacttaa ataacattta ttctgcgatt gttggataca gtgttctata tttgtcaatt 36240 agattgagtg ctgctcagat gttctatatt cctacagata tttcatgtgc ttattctata 36300 aaatacaaat tgttaatgtt tctgattagg actagaagac ttttctttta ccttttaaat 36360 atatttttta ttttatgtat tttgaaacta tgttatttgg tgcctataca cttagatttt 36420 tttcttgagg aattgatcct ttatcattat gaaatatcct tcaggcaccc cgctcccaca 36480 gatgtatgca tcttgctgta cccctgcttc tgctggcaca aatgcacagg catggatcct 36540 gctgccacca ccttgatgaa gtgtgtggcc agcaccccca tcagagtact gttaccagca 36600 tactgggaat accttgcccc cagagtgcag cagctttcta acctctatgg ggcagagaac 36660 aaagttgggg ggcccagtac cagcccatca gcgttacatc acatagccca tgagtgatga 36720 gctgagcctt ggtgccctga aagcatccag aatgaagcca gtcaactgaa ccgacattat 36780 accacaatca aaccctcaag aatatcaaag aatataaaag taaaaagccc tatccaaagg 36840 atagcaactt caaagattaa aggaatgtca gcccacacag atgagaaaga atcaatgcaa 36900 gaactctggc aattcaaaaa actagagtgc cttcttacct ccagacaacc ccactggttc 36960 cctagcaatg gttcttaaca aaactgaaat gacagagata gaattgagaa tctggataga 37020 aaaaagatca ttgagattca ggagaaagtt gaaacccaat ccaaggaatc taaggaatcc 37080 aataaaacaa tacaagagat gaaacgaaat agccatttta agaaagaacc aaactgaact 37140 gatagagctg aaaaactcac tacaagaatt tcataatgca atcacaagca ttgttaacat 37200 cagaatagac tactaacatc agaatagacc aagctgagga aagaatctca gagcttgaag 37260 actggttcct caaatcaact cagacaaaaa taatgaacaa ataattaaaa agagtgaaca 37320 aaaccactga gaaatatgag attaggtaga gaccaaatct gtgactcatt gacatccctg 37380 aaagagagag agaaaaagca atttttccag ccttgctaga gaggccaaaa ttcaaattca 37440 ggaaatgcag aaaacccctg tgagatacta tacaagacaa ccatccccaa gacatgtagg 37500 catcagattc tccaacgtca acatcaaaga aaaaatacta aggcagctag agaaaagggg 37560 caggtcacct acaaagggat tccgatgagg ctaacagaag acctttcagc aaaagcacta 37620 caagccagga gagatttgga gacctatgtt cagcaaaaaa aattccaacc aagtatttcc 37680 tgtgcagcaa gactaagctt aataagcaaa ggagaaacaa gatccttttc agacaagcaa 37740 atgctaagag aattcattac caccagacat gccttacaag aggtccttaa gtgagtgcca 37800 aatatggaaa caaaagactg ttactggcca ccacaaaaat atacttaagg actagacctc 37860 tgaacactat aaagcaatta cgctatcaag tctgcataat atccagctga catcatgatg 37920 acaggatcaa atcaacacat ataagtatta tccttaaaag taaacatgca aaatgcctga 37980 cttaaaaagt acagaatggc aaatttgata aagaaataag acacatggag agttgctcca 38040 aatggcagga taggaacagt tctggtctac agctcccagc aagatcaatg cagaagacag 38100 gtaatttctg catttccaac tgaggtacct ggttcatctc attgggattg gttggacagt 38160 gggtgcagcc catggagggc aagccgaagc agggcggtgc atccctcacc gaggaagtgc 38220 aaggggtcag gggatttccc tttcctatcc aagggaagcc atgagtgact gtacctagag 38280 gaaaagtaca ctcctgctca aatactgcac ttttcccacg gtattcgcaa caggcagacc 38340 ggaagattcc ctcccatgcc tgctcggtgg gtcccacgct catggtgcct tgctcactgc 38400 tagcacagca gtctgagatc gacctgggat gctggagctt ggtgggtggg gagcggcgtc 38460 caccattgct gaggcttgag taagcggttc tatgctcaca gcgtaaacaa agcagcaggg 38520 aagcttgaac tgcacggagc ccactgcagc tcagcaaggc ctactgcctc tgtagattct 38580 acctcggggg cagggcatat ctgaaaaaaa tgccgcagac agcttctgca gacttaaaca 38640 tccctgcctg acagctctga agaaagcagt ggttctccca gcacgggggc gttcaagctc 38700 tgataacgga cagactacct cctcaagtgg gtccctgacc cctgtgtagc ctgattgggg 38760 gatacctccc agtaggggcc aacagacatc tcatacaggc gggtgcccct ctaggacgaa 38820 gcttccagag gaaggatcag gcagcaatat ctgctgttct gcagcctcca ttggtgatac 38880 cgaggcaaac ggtctggagt ggaactccag caaactccaa cagacctgca gctgagggga 38940 ctgtctgtta gaaggaaaac taacaaagag gaatagcatc aacatcagca aaaaggacat 39000 ccacacgaaa accccatccg taggtcacca acatcaaaga ccaaaggtag ataaaaccac 39060 aaagatgggg agaaaccaga gaagaaaggc tgaaaattcc aaaaccagaa cgcctttctt 39120 ctccaaggat cacaactcct cgccagcaag ggaacaaaac tggacagaga atgaatttga 39180 tgagttgaca gaagtaggct tcagaaggtc ggtaataaca aacttctcca agctaaagga 39240 gcatgttcta accaatcgcg aggaagctaa aaaccttgaa aaaatgctag atgaatggct 39300 aactagaata caataaccag tgtagagaag aatataaatg acatgataga gcttaaaacc 39360 atagtacaag aactttatga aacatacaca agcttcaata gctgattcaa tcaagcgaaa 39420 gaaaggatat cagtgattga agatcaaatt aatgaaataa agcaagaaga caagattaga 39480 gaaaaaagag tgaaaagaaa cgaataaagc ctccaagaaa tacgggacta tgtgaaaaga 39540 ccaaatctac gtttgattgg tgtacctgaa agtgacaggg agaatggaac caggttataa 39600 aacactttca ggatattatg caggagaact tccccaacct agcaaggcag gccaacattc 39660 aaattcagga aagacagaga acaccacaaa gatactcctc gagaagagca accccaagac 39720 acataattgt cagattcacc aaggttgaaa tgaaggaaaa aatgttaagg gcagccagag 39780 agaaaggtcg ggttacccac aaagggaagc ccatcagact aacagcagat ctctcagcag 39840 aaacactaca agccagaaga gagagggggc cgatattcaa catgcttaaa gaaaagaatt 39900 tttaacccag aatttcatat ccagccaaac taagcttcat aagtgaagga gaaataaaat 39960 cctttacaga caagcaaatg ctgagagatt ttgtcaccac caggcctgcc ttacaaaagc 40020 tcctgaagga agaactaaac atggaaagga acaaccggta ccagccacta caaaaacatg 40080 acaaattgta aagaccatcg atgctatgaa gaaactgcat caattgatgg gcaaaataac 40140 cagctaacat cataatgaca ggatcaaatt cacacataac aatattaacc ttcaatgtaa 40200 gtgggctaaa tgccccaatt aaaagacaca gactggcaaa ttggataaag agtcaagacc 40260 cttgactgta ttcaggagac ctatctcaca tgcaaagaca cacataggct caaataaagt 40320 gatggaagaa gatctaccaa gcaaatggaa agcaaaaaaa agggtttgcc atcctggtct 40380 ctgatgaaac agactttaaa gcaacaaaga tcaaaagaga caaagaaggc cgttaacata 40440 atggtaaagg gatcaattca acaagaagag ctaactatcc taaatatata tgcacccaat 40500 acaggagcac ccagattcat aaagcaagtt cttagagact aagtctacaa agagacttag 40560 actcccacac aataataatg ggagacttta acaccccact gtcaatatta gatggatcaa 40620 cgagacagaa agttaacaag gatatccagg acttgaactc agctctagag caagcagaac 40680 taatagacat ctacagaact cttcttccca aatcaacaga atatacattc tcctcagccc 40740 cacatcacac ttattctaaa attgaccaca taattggaag caaaacactg ctcagcaaat 40800 ataaaagaac agaaattaca tcaaactgtc tctcagaccg caatgcaatc aaattagaac 40860 tcaggattaa gaaactcact caaaactgca caactgcatg gaaactgaac aacctgctcc 40920 tgaatgacta ctgggtaaat aacgaaatga aggcataaat aacaatgttc tgtgaagcca 40980 ataagaacaa agatacaacg ttcaagaatc tctgagaaac atttaaagca gtgtgtagag 41040 agaaatttac agcactaagt gtccacagga gaaaacagga aagatctaaa atcgacaccc 41100 taacattgca attataaaag aactacagaa gcaagagcaa atgaattcaa aagctagcag 41160 aagacaagaa ataaatcaga gcagaactaa aggagagaga gacacaaaaa acccttaaaa 41220 aaaatcaatg aacccaggag ctggttttct gaaaagagca acaaaataat tacatgacaa 41280 gcaagactaa taaggaagaa aagagagaag aatcaaatag acgcaataaa aaatgataaa 41340 ggggatatca ccaccgatcc cacggaaata caaactatca tcagacaata ctataaacac 41400 ctctacgcaa ataaaataga aaatctagaa gaaatggaaa aattccttga cacatacacc 41460 ctcccaagac taaaccagga agaagttgaa tctctaaata gttcagtgac agcttctgaa 41520 attgaggcaa taattaatag ccaaccaacc aaaaaaagtc caggaccaga cagactcaca 41580 gccaaattct accaggggca caaagaggag ctagtaccat tccttctgaa actattccaa 41640 tcaatagaaa aagagggaat cttccctaac tcattttata aggccaacat catcctgata 41700 acaaagcctc acagagacac aacaaaaaaa gataatttta ggccaacatc cctgatgaac 41760 atcgatatga aaattttcaa aaaaaaatac tggcaaaccg aatccagcag cccatcaaaa 41820 agcttatcca ctacgatcaa gtcgacttca tccctgggat ggaaggctgg ttcaacatag 41880 gcaaatcaat aaatgtaatc catcacataa acagaaccaa cgacaaaaac cacatgatta 41940 tctcaataca tgcagcaaag gccttcaaca aaattcaaca gcctttcatg ctaaaaactc 42000 tcaataaact aggtattgat agaatgtatc tcaaaacagt aagagttatt tatgacacac 42060 ccacagccaa tatactgaat gggcaaaaac tggaagcatt ccctttgaaa actggcacaa 42120 gacaaggatg ctctctctca ccactcctat tcaacatcgt gttggaagtt ctggccaggg 42180 caataaggca agagaaataa ataaatggta ttcaattagg aaaagaggaa gtcaaatagt 42240 ctgtttgtag atgacatgat tatatatgta gaaaacctca tcgtctcagc tccaaatctc 42300 cttaagctga taaacaactt cagcaaagta tcaggataca aaatcaatgt acaaaaatca 42360 caagcattcc tatccaccaa gaacagacaa acagagagcc aaatcatgag tgaactccca 42420 ttcccaactg cctcaaagag aataaaatac ctaggaatcc aacttacaag ggatgtgaag 42480 gacctcttca aggagaacta caaaccactg ctcaatgaaa taaaagagga tacaaacaaa 42540 tggaagaaca ttccatgctc atggataggg agaatcacta tcatgaaaat ggccatactg 42600 cccaaggtaa tttaaagatt caatgctatc cctatcaagc tatcactgac ttcacagaat 42660 tggaaaaaaa ctactttaaa gctcatatgg aaccaaaaaa gagcccacat agccaagaca 42720 atcctgggca aaaagaacaa agctggaggc atcacactac ctgacttcaa actacactac 42780 aaggctacag taaccaaaac agcatgttac tggtaccaaa acagacatgt agaccaacgg 42840 aatagaacag aggcctcaga aataacacca cacatctaca accatctgat atttgacaaa 42900 cctgacaaaa acaaatcggg aaaggattcc ctatttaata aatggtgctg ggaaaacagg 42960 ctagccatat gtagaaagct gaaactggat cccttcctta caccttatac aaaaattaac 43020 tcaagatgga taaaagactt gaatgtaata cctaaaacca taaaaaccct acaaaaaaac 43080 ctaggcaata cccttcagtg cataggcatg ggcaaagact ttatgactaa aacaccaaaa 43140 gcagtggcaa caaaagccaa aattgacaaa tggcatctaa ttaaactaaa cagcttctgc 43200 acagcaaaat aaactaccat cagagtgaat aggcaaccta cagaatggga gaaaaatttt 43260 tcaatctatc catctgacaa agggctaata ttcagaatgt acaaagaacc taaacaaatt 43320 tacaagaaaa aaaaaaccac tgcatcaaaa agtgggcaaa ggatatgaac agacacttct 43380 caaaagaaga cagttatgca gccaacagac atatgaaaaa atgtccatca tcactggtca 43440 tcagagaaat gcaagtcaaa accacaatga gataccatct catgccagtt agaatggcga 43500 tcattaaaaa gccaggaaac cacagatgct ggagaggatg tggagaaata ggaacacttt 43560 tacactgttg gtgggagtgt aaattagtgc aaccattgtg gaagacagtg tggcgattcc 43620 tcaaggatct acaactataa ataccatttg acccagcaat cccatttgtg ggtatatacc 43680 caaaggatta taaagcattc tactataagg acacatgcac acatatgttt attgcagcac 43740 tatttacaat agcaaagact tggaaccaac ccagatgtcc atcaataata gactggatat 43800 atagtattct atgtctggtt aaaaaaaaag ttattcaatt ttatggattt ggattaggat 43860 cattcccatt tctgtaaata tttacaccta taaggatatc aagaaggaat gtcaggcagt 43920 ttactagtaa cttttcaaag tcttagaaaa gttgcatttt ttgctggcac caagagggga 43980 aaacatcata aaaatagttt caaaatctac aatcaatgct ttctccaaaa taaatgtctt 44040 tgttaaaaaa aataataata atagactgga taaagaaaat gtggcacata tataccatgg 44100 aatactatgc agccataaaa aaggatgagt tcatatcctt tgtaggtaca tggatgaagc 44160 tggaaatcat cgttctcagc aaactatcac aaggacagaa aaccaaacac cgcatgttct 44220 cactcataag tgggagctga actatgggaa cacgtggaca cagagagggg aacatcacac 44280 accggggcct gtcagagggt gggggactgg gggagaggta gtgttaggag aaatacctaa 44340 tgtaaatgac gagttgatgg gtgcagcata ccagcatggc acatgtataa ccacgtaaca 44400 aacctgcatg ttgtgcacat gtacccttga atttaaagta taatttaaaa aaagaagaag 44460 aaataagtca caattgtatg ctgtcttcaa gagacccatc tcatgcagag ctacccatag 44520 gctcaaaata aaggtttaaa gaaaaatcta ccaagtaaat gggaaaagaa aacaggagtt 44580 actattctaa ttttagacaa agcagacttt aaagcaacaa tgatcaaaaa agacaagcgc 44640 atgacataat cataaagggt tcaattcaac aagaagactt aaatatccta aatatatgta 44700 cccaacacag gagcttgcag attcatagaa caaattctta gagacctaca aagagacatg 44760 gataaccaca cggtaatatt gggagacttc agcaccccac taacagtatt caatcatcaa 44820 ggcagaacac taacaaagat acttgggacc tgaactcagt atttgactaa atggacccaa 44880 cagacatcta cagaactctg cactccaaaa caacagaata tacattcttc tcatcgccac 44940 atggcacata ctctaaagtc gaccacacaa tcggccataa gtcaattctt agcaaattaa 45000 aaacacaaga atcacactaa ccacactctt ggaccatggc gcaacaaaag tagaaatcag 45060 ccccaagaag atcattcaaa acatacagct acatgcaaat caaacaacct gctcctgaat 45120 gacttttggg taaacaatga aattaaggca gaaatcaaga cattttttga aactattgaa 45180 aataaagata aaatatacga aaatatctga gacacagcta aaataaagat aaaatatacc 45240 aaaatctctg cgacacagct aaagcagtat taagagggaa gtttatagca ctaaacaccc 45300 ctatcaaaaa gttagaaaga tctcaaatta acagcctaac attgcaccta gaggaactag 45360 aaaaacaaga gcaaaccaac tgcaaagcta gcagaagaaa agaaccaaaa caagaactga 45420 actgaatgaa attgagacag gaaaatgata taaaagacca gtgaaaccag aagctggttc 45480 tttgaaacaa taaataagat ggatagacca ctgattatac taatcaaaaa acagagcaga 45540 tccaaataaa cttaatcaga aatgacaaag aggacattac catcaaccca acagaaatat 45600 aaaaaacctt aaaagactat tacaaacacc tctatgcaga caaactaaaa aacctacaag 45660 aaacgataaa ttcttggaaa catacagtct cccaagactg aacaaggaag aaattgaaac 45720 cctgaacaga ccaataacaa gttccaaaat tgaatcagta gtaaaaagcc taccaaccaa 45780 aaaagccctg gaccagatgg atccacaacc aaattctgcc agatgtataa agaagagctg 45840 gtaccattcc tactgaaact attccaaaaa attaaggagg agggacccct ccctaactca 45900 ttctataagg ccagcatcat cctgatataa ggccagcatc atcctgatat aaggccagct 45960 tcatcctgat aacgaaaact gggagagaca acataaaaag aaaacctcaa gtcaatatcc 46020 tggacaaaca tagatgcaaa aattctcaat aaaatactag caatctgaat tcagcagcac 46080 atcaaaaaac taatccacca tgatccaagt aggctttata cttggcatgt gagattggtt 46140 caccatatac aaatcaataa atatgattca tcacatgaac agaactaaaa agaaaaacca 46200 aatgataatc tcaatagatg cagatacatc tattgaatgc aaaattcagt accccttcat 46260 gttagaaacc ctcaacaaac taggcatcaa aggaacatac ctcaaaataa taagagctat 46320 ctgtgaaaaa atcacagcca gcatcatact gaaagagcaa aagctggaag cattcccctt 46380 gtggaataag acaaggatgc ccactatcac cactcatatt caatatacta ctggaagtcc 46440 taggcagagc agtcaagcaa gacaaataaa gaaaaggcat ccaaatagga agagaggaag 46500 tcagacaata tgattttata cctagaaaat tccatagtgt ctgcccaaaa gctcctagat 46560 ctgacaaaca acttcagcaa agttgcagtg tacaaaatca ctgtgcaaaa ataagttgca 46620 ttcctttaca ctaacaacat ccaagctgag agccaatcaa ggacacaatc ccattcataa 46680 tagccacaca aaaaataaaa tacctacgca tacagctaac cagagaggca aaatatttct 46740 agattgagaa ttacaaaaca gtgctgaaag aaatcagaca acacaaacaa acgagaaaac 46800 attcattcca tgctcgtgga taggaagaat caatgttgtt aaagtggcca tactgcccaa 46860 agcaatttac agattcaatg ctattccttt caaactacca gtgatatttt tcacagaatt 46920 agaaaaaact attctaaaat tcaggtggaa ccaaaaaaga gcctgaatag ccaaatcaat 46980 cctgagcaaa aagaacaaaa ctggaggcat cacattacca aaattcaaac tgtactgcaa 47040 gcttacagta acaaaaacag catggtactt gtacaaaaac agacacataa accaatggaa 47100 tagagagccc agaaataaag ctgtgtactc agaaccatct gatctttgac aaaattgaca 47160 aaaacaaaca atggggaaag gacctgctag tcaataagtg attcgggatg gatcgctacc 47220 catatgcaga agattaaaac tagaccactt cctatcacca tatataaaat caactcaagg 47280 atgtattaaa gacttaactg taaaaactat aaaaccctag aggaaaacct aggaaatacc 47340 attctggaca tagtccctgg caaagatttc atgatgaaga tgccaaaagc aattgcaaca 47400 aaaaaacggc aagtgggacc taattaaaga tcttctgcac aacaaaaaga aactatcaag 47460 agagtaaaca gacaacctat agaatgggag aaagtatttg taaagtatgc atctgacaaa 47520 ggtctaatat ccagaatcta taaggaactt aaacaagttt acaagaaaaa aaaattttta 47580 aagtagctaa agtaaatgaa cagacacttt tcaaaagatg acatacatgt gaccaacaag 47640 catatgaaaa aatgcccaac atcactaatc attagagaaa tacaaattaa aaccacagtg 47700 agataccatc tcacaccagt cagaatgggt attattaaaa agttataaaa tagcagatgt 47760 tgacgaggtt atggagaaaa gggaatgcct atacgctgct ggtggaaatg taaattagtt 47820 cagccattgt ggaaaacagg gtggcaattt ctcaaagtac ttaaaacaga actaccattt 47880 tacccagcca tccacttatt gggtatatgc ccaaaggaat agaaatcatt ctaccataaa 47940 gacacatgca cacacacgtt catcgcagca ctattcacag taggaaagac atggaaccaa 48000 cttaaatttc catcactggt agattggata aagaaaatgt ggtacatata caccatgtaa 48060 tactacgtaa ccataaaaaa agaataagat gatgtccttt gcagcaacat ggatagtgct 48120 ggaggtcaat actctaagcg aactaatgta ggaacagaaa accaaatgct gcatgttcta 48180 acttataagt ggaagctaaa cattgagaac acatggatac aaagaaggga ataatggaca 48240 ctggacctac ttgagggtgg agggtaggag gaggatgagg agagaaaaaa tgacctaata 48300 tgcttattac ctgggtgatg aaaatctgta caccaaaccc ccacaacatg caatttattg 48360 ataacaaacc tgcacatgga cccctgaacc taaaataaag tcttaaaaaa agaagagggg 48420 ccgggcacag tggctcacgc ctgtaatccc agcactttgg aaggctgagg cgggtggatc 48480 aggaggtcag gagattgaga ccatcctggc caacacggtg aaaccccatc tctactaaaa 48540 atacaaaaaa ttagccgggt gtggtggtgg gcttttgcag tcccagctac tcgggagact 48600 gaggcaggag aatggcatga actcgggagg tggagcttgc agtgagctga gatcatgcca 48660 ctgcactcca gcctgggcaa caaagcaaga ctccgtctca aaaacaaaag aaaaacaaaa 48720 aaacaaagag gaggaagagg aaaataaggc ccatggtgct taaagtcaca catgtgccaa 48780 agccagctag taaacaaagc aaagatctga attcagatat ttgtttaact gctgtggttc 48840 taattatatc acgcaccttg gactcaaagc aggatagaga aggttgggca gtggatcaca 48900 aaagatatca gcataaatac acatcattta ttctgtatta tctgatgcca agcagtgtgc 48960 taagcacttt acatacatca tcttatttgt acatcccaat taccttctga tacagctgtc 49020 aattcccact tttatcagga aactgaggat caaagagggt aagtaataca tccagagtta 49080 caaagctgtt aaaaatatta gagggtctga ctccagtgta tatattttta aaagcccctt 49140 attatctctg gtaatcactg tcttgaaaac tactttgtct gatattaata aagccacagt 49200 agctttcata caccttcttg tttacatatg atttcccata ttcttttatc ccatctatgt 49260 tattaaaatg tgtcttgtca atagtatata gtttgttctt gtttctttat tcattttgtt 49320 ttcctgatac tcttttccca attaaggagg taataatctg taagcaaagc cttatttaca 49380 taaacactgc tgtgatctta taccaaataa acagccctag catgtgtgtg tgtgtgtgag 49440 agagagagag tgtgtgaatg tgtgtgtgtt tgggctttct gaattagatt ttctttcaag 49500 atccacctac atctttactt ttggcctaag taatggttgg tcatgttaac tgtgtaaaat 49560 attaatcttt ttctgaagct acaaagagtc catgtctact tactgttcta ggaaaagttt 49620 tttcaaattt gggaagtgtc taccttttaa aatattttct gagagtgttt aattacttct 49680 atgatcttaa aaggttctct ccattttact tttatccagc tgtcacatat tctgagtatg 49740 ttttccacca ggagaacctg agaatttttt tttctcttag attttgagta tagagataag 49800 attttatcct gcatccagtt ccaacctgtt aatataattc tatgcaagtt tcctctcttg 49860 ttgtgtttat ttccctttat ccttatcctt agtcatctat tccaatgttt atgctgtgca 49920 ttattatatt ttcatgtatc cttgtgaaat gtagagtgct ttatgtgcaa tattttaaat 49980 gtacataaat ggcattatag attaaaacat tttttcatgt tgtaccatgt ttttgaggtt 50040 tatttatatt gctatgtata cctctagtct actgcttcta actgtgaaac atttttttca 50100 aattcaaatt ctcataaatg ctgtacttat tttggaaatt agaattcagc acaaggtact 50160 tctctgtatt caacaccata gcattttagc gctttactaa tgggtggaaa aattaaactt 50220 cagtcctact gttcaaagag cctatttagt caaagatggc caagatggaa agcttccact 50280 ttcctgtgag tcaggtccta gtaaatgtga atcttacaca gagatgatta gttatagctc 50340 ctagcactaa ctccctggag gcaatgtcct tggtgcccct gactctcttc catttaggca 50400 ctcatctttg ttagatgcct taggtttatt tacgtaaaat catgctgcac ttctccctga 50460 tactgtggtt aaagttttta tggccttgca ccaaaccaac agcccagaat agctacatgc 50520 cagggagata ttgtcagcaa ggtttgggca ttaatatgaa gaatcttgta tattgttgaa 50580 acttctggtc caacagaatg gaaagtgtgc caaataggaa gcttttgggt acaggtgaca 50640 aaaaccccaa ctcaaactgg cttcaacgct aagcacatat attatctcac tatattagca 50700 gaataactaa atattggcag ctgcagaggc aaaccctgac tactgagagg cttgatacag 50760 taaaagctta ttgcttggtc tcacaaagtt tgccaggggt tggtgactct cctgggcagt 50820 tagtgactca gatatgtcgc tttataagca acacatgact tgtaaagtca cccctgcaag 50880 ggaagaggac tggtggatca catagattgt ttttaaggac caggctggaa attgacttat 50940 atgaattttg cccatgctcc agtgtgcata aatccgtcat cagtcatatg accccatcct 51000 aactgcaaag gatgcagaaa catggagtct tcctgtgaac ttaggaagag gaaaagtgtt 51060 gagcaagaag ccacattcca taacaggaag tccagatata cagcaggttt tagggatgga 51120 tgacttagtg actcagtggt gtcatcaccg tcctgaagtc tttccaatgc tctccaccct 51180 gccgtcctca caatgtcagc tttgtcttca ggctggttct cttcctggtt gcaaggtggc 51240 tgctgtagtt cagccatcac aaccagacat ggcaatgtgc tgggacagaa agggaccatc 51300 cttaggagtg agggcacttt tcccagaagc agcccagcac acttcctctg atggccagga 51360 ctgccaggcc caaactaatc actgacaact caagatttat ccctggcatt gagagaccat 51420 caccctcttg gcagtcatgt gggggaaggg ctggttacca tttgaaccaa aatgaagctt 51480 agccaataag gaagagaaga aagtagatgt tgagtaggca acgaacagta tctgctatag 51540 cccatccctg gactcgctga ggtttcatgg taaagtttgt ggttttcatc atgttcctgc 51600 accattctca gtaagttgat gcctagcatt gcatattttc ttttgctatt gtaatgctgt 51660 ttccatgata gttgcttgtg tataggattg tcattgattt ttcactcacc tttgttcagt 51720 tttcttttat ctatgtgtct gtgtaattgc cttaaattct tttttcaagc agtattagat 51780 gttaattaga gactgagaaa cccagaggtg gaaggatctt aagtggagca ggggatcgga 51840 ttaaagaata agaaagcaaa gaatactgta tttcatttct gtgaaaagag aggttacatt 51900 tccttgcatg aggaaaaagt aggagtgtgt gcttttctca aaacttgcta taaggctggg 51960 tgcagtggct catgcctata atcagcactt taggtggcca aggcaggagg attgcttgag 52020 cccaggagtt tgagaccagc ctgggaaatc tagtaaagcc ctgtatctac aaaaaataaa 52080 aaattagctg agagtggtgg tgcacgcctg cagtccccag ctagctggga ggctgaggca 52140 ggaggattgc ttgagcccag gaggaggtca agcttgcagt gagctgtgat catgccactg 52200 aactccagtc tgggtgacag agtgagactc tgtcttaaaa aaaaattgct atgagagaat 52260 gctctaggta tgtgtgtggt cgtggtagag tataggggtg tacgatcact gtaatgtgct 52320 ggaatagtac ctaattttga aactgcatgg ttaataacat cttttaagaa tgtctaagtg 52380 ctttagagac ttcttttccc ccattctccg tgagaagaat aggagataga tcgttggttt 52440 tcgtgtggaa aaattaagaa agagaaatga agcagtttaa atttcctcac attatcatgg 52500 tattaagtaa actagagcag aggtctttgg gttagaactc atttgggaaa atagattgag 52560 tgtggccaag gcaaccattc ctgtagtgaa ctcagctccc ttttatgtag tgaagataaa 52620 tagttgaaca atccaataca tgccccagtc cctctctctc tacacacact gcacacacac 52680 acacatacac acacacacac acacagtgcc aggagacatt ccgggaccat atttttttaa 52740 gaagaaaaag tattattatg ctttcaagtc agttatgtta atttattcct tttttgagat 52800 gtagagtagc tcctttgtat ctgagtttgc ctattacatg aaaagactag tggataatga 52860 taccagctat cctatgttac aaaaagctgc agatccccat gccaagtgga ctatacagct 52920 acacaaacct tctgttacct gttggacaca ctgccacaga ggcgagcaac atgctgtctg 52980 ccattaaaga actcgggacc cttgaaaccc attttgggga atatatgtag caaataatcc 53040 aattttaaaa ggggcaaaaa cgtaggttat cggagtacat tagacatgct aatctgcagc 53100 agttttgtgc aaacctaagt ataatttaat tttgtattaa gttttccttt tgatatccag 53160 ataaagatta acatagtcat tggcaattat atttgtattt tcaataaaca tcacatttta 53220 ctgaagtata cagatctatg caagggatag tgaaatagac aaagctcatt tgacttttcc 53280 agaaggaatg cattttaaca ttggtggctc ctgtggttag ggggacttcc aaagcagtaa 53340 ctcttgcgtt tcctcagcag catcatcata attacaccac tatacacaat aattatactc 53400 ctagctgtgg ttctattcta cagcagttgc tgctttcctg gaaaccttgg ggaaaataga 53460 aatttaaata gctcatagat ttggctatga taagttcaca caaagacaaa attgcatttg 53520 gcagtttagt ttccccaacc tttatttgtt tcctgataac tatatttagt ctgaacctct 53580 ttgtcaagga caccacatag gttatattgt cttcctcttt gcatccaatg aagcagcata 53640 tattgccagg tcattttttt ttatggtgat gccaaacttg accacttggc caagggagtg 53700 actgccatac ctcttattgt gaaggcacat tttcctcctg taattaacaa gtaaccccag 53760 gggtgatacc ttgagacaat gagaatattt tgttttccaa taacttttac ccaagaatag 53820 ctacacacac cccctgtaat tatagactta tgggtcagtt tttctcacca tgtgttttaa 53880 tccatcgatg tcattctttc tgatatttaa aatcgttcac agattggcca ggaacccttc 53940 agaccagctt ctgtcttcat tgaacttgtc cttgtttagc tttgagcact tcttggcgtt 54000 tttggcccaa gatatcttag gcttatctcg tactttcagt actctggacg tgtgtcagac 54060 ctgaaatcag cctcttctcc aaggagacct gtttcccttt agtggaaaat aatatttaca 54120 aaccaagagt tgggtgtttg tgttttactg ctactggggt gttaattttt ctaggctctt 54180 taatcgtgag ttcatattga aaccacctaa ttattttgtt tttattttga attacctttc 54240 ttgaggtata gtttacctaa acgaaagtgt actcatttga agtgctgagt ttgcttcatg 54300 ttagcaaatt catataccct gaactgtcac cctgtcacct atgacctttt gcagtcagcc 54360 ctctttcagc ctctgacctg ctttgtgtca ctgtagatta gtgtacctat tctagaattt 54420 catgtaagtg gaataacact atatgaggcc ttttctctgg ctacttttac tcagcattat 54480 gttttggaaa ttggtacatg gtattatgca taccaattat tcattactgt ctcttactga 54540 atagcagtcc attgtatgta tgttttgctt atctatttgt ttatctattt acaagttgat 54600 gaacatttgg accatttcca gcttatggat attatgagtg aaactgtttg aacattcagg 54660 cacaaggttt tgtatggaca tgagctttca tttctctgca gtaaatacct aggaaaggaa 54720 ttgctgtgtc atgtttttta actttatagg aaactaccaa ttttttttcc caagtagttg 54780 ttaccatttt ttactaccac caagaatgta tatgagacct aattattcca cattgtcatc 54840 agctcttggt attgccaatc ttttcatttt agccattctg gtgggtcagt agtggcttta 54900 atttctgttt tttaattttg tttaatttct gaaattttaa ttttattgtg gttttaattt 54960 ccatttctgt aatgggtgat gacacagagc atctttttgt gtgataattg gtcattcata 55020 tatcttgatg aaatgtccat tcatatcttt cactgacttt tttaattggg ttgtcttttt 55080 tcttgagcta tgaagttcta tgcattctga atactagtgt tttgtcagat gtatgtatta 55140 ggatatgttc tcccagtcta cagattactt taaaaatttt cctaatagta tcttttggag 55200 agtagaaaat tttaattttg atgaagtcca atttatagat ttatttaagg ttcctattat 55260 ttatgtcgtg tttaaggaag ttttgcctac cctaagacct caaagatttt ctcctgtatt 55320 ttcttctaga agtttgtaaa cttaaaaact ttagcttttg tgtttgggtt catggtccca 55380 ttttaattta atttttatga attttgtgag atataggtgt tgttcttttt ctttcttttt 55440 taatttttta gttaataaaa atggtatata tttatattgt ataacatgat gatttgatac 55500 atgtatacat tgtggaatac atattacctc acatggttat catttttttg tgataagaac 55560 acttaaaaat ctgtctcagc aatttttaac tatataatat attaactata gtcaccatga 55620 tatacaatag atctcttgaa cttattcctt ctaagtaaaa atttgtcctt tgaccaccag 55680 cccccctgcc acccctcact ccctgtccaa cctctgatac caccatttta ctttctgttt 55740 ctatgagttt gactttttaa gattccattt atcagtgaga ccattggtat ttgtgtttct 55800 tagcctggtg aattttactt agcataatgt cttcccagtt tttttatgtt gttgcaaatt 55860 acaggatttc cttcttttct aaggcagaat agtaccctat tgtgtttatg tgtatatata 55920 tatatgtgtg tgtgtgtgtg tgtgtgtgtg tgtatatata tatatatata tcccattttc 55980 ttaatccatt catctgttga taggcactta ggttgatttt gtatcttggc tattgtgaat 56040 agtgctgcaa taaacatggg cattctcttt gacatactga tttcatatcc tttggatata 56100 tatgttcaat aatggaattg ctgcatcatt tggtagctct atttttaata ttttgaagag 56160 cctccatact gttctctaca atggctatgc caatttacat ttctaccaac catgtacaag 56220 ggtccctctt tctccacatc tacttttttt ttttagcatg gatgtccatt cattccagcc 56280 ctgattgctg aagagactgt ccttttcccc cattgaactg ccttggcatc ttagccagaa 56340 atcgactgac tatatgtagg aggaactatt tctaaattct ctagtctctt ccatttatct 56400 atgtatttat ccttacacta atgccacatt gtcttgattt atatagcttt ataataagca 56460 gagcccagtc aggcccctcc tagacagtcc ataccagaat tcacaaatac atatgaccta 56520 aagcaagtaa cataattaag tgaagctgtt cttgtataag cagtattact ttatttatat 56580 taaaatctaa cgggctaaaa tttcaacgtg gttcattttc atttattaat gttttgagtc 56640 atgtcccttt ctcacatgtc tcatggttct tattatggtt tatttctaat gatatcttta 56700 tacacatact tttcacacta aatatttagg aacattccct gttttcatac atagaacaat 56760 actgcctcca tacatttttg aaatgcgtgg cccatttgat ctgtttttca gtttcccact 56820 tcaaagagag gaatacagta tctcccatga cagcatcagc tggttaatga atggtgattg 56880 gcatgcagac tcagcattga gactgcagtg gggaatgatg gggacactgt ggcaaacggg 56940 ggagggctgg cactgctgag aggggcacag ccactctacg ccactttcat aacatcatgt 57000 caaatgtaca caaattcacc attatttatt ttgttgctca aattattcca gctttgtcca 57060 ctaggagctg tttcaattgg ctatgtccct ttgacacaca caccaatgtg ggtttgttcg 57120 gttttttgtc ttatttgaat gcttctttcc tttctggctc ctccaggctt atcttgtgta 57180 tttcccatcc cagtcctaga atcagccatt tctctaagaa tccctggttt cttttataag 57240 agaatggcat tagaaaatgc cacagaggtt aagtgtcatt cccatcacat catagcaagg 57300 gtacatacta tcaacacgat ttatgactat cgatgttacc cttaatcatc tagccaagat 57360 ggatggtgaa atcattagga gggggatgca ataaagagga agaaaagcag actttatgtt 57420 cattttcaag cttgttccaa aggaaagtta taggtgtatt tgatcatata aatatcgttt 57480 tgttttggaa aatcacgttt agggacaata aaattggaaa gaatccttgc ttccaaatca 57540 cctctgtgtt caaatcttca tcatatttgc actgcccttt cctttgtgtt cttagttcat 57600 gggtcccatt ggatttgatg ggtttcgttt actttttgct actgaaattg ttagtttaaa 57660 aaaacaggat acagagtgtc acatttttta ttgtaactcc actttcttga agttgcctat 57720 agcattttaa atttcagaag atcgataact ttattcattt ccctttggaa aaaataaaac 57780 aagattttaa ctagtggcat gtcttcaagg aagctaaaga catttaactt tttctgtttt 57840 gctctaaggg cattctgatg attgcctgaa tgtgatcttc tgagtgttcc agtccctgga 57900 gctggctgtt agccatctcc caaggagggt cgaaaagaac gtagcagaaa gctgcagaaa 57960 gaaaagctgg ctaaagaaac aggagctgcc agccagggca acatagtgag accccaactc 58020 tatagaaaat ttaaaaagct gtcctggcat ggcatgcctg taatcttagc tactcagaag 58080 gctgaggtgg gaggatcact tgatcccagg agttgggggc tgcagtgagt acttccagcc 58140 tgggtgacag aggagaccct gactcaaaaa aaaaaaaaag agccggttgt tgttaagggc 58200 caccagactg gagtgcatgg tagtcacctg cctccctcct ttcctggaga agatgacaac 58260 gtgatgaaca agcaccacta ccctggaata gtttaaatgg cattcagcct ggaggagtgt 58320 gcacatgcac caaatatatc ttgtgtgtcc tttgtagcca ttgggtctat ggagattact 58380 ctggctgtga gtttggttga ttaacattgc tatgcaagcc ctgtccattt agctggaggt 58440 tatcacagga ggaataaacc acagaatatg actttggcat tgaataaatg ggaagagagt 58500 gcttttgggg gccagaactg tttgtaatat tataactcct ataaatacat gaattgctgc 58560 ccctttatca taatatccat tgctttaatg ctctgtttta gattggagtt cacaccagac 58620 acctgagatt gtgtttgtta tgtaactgct atttcatgtt gagaaagtag caaagctttt 58680 aaatactgag aatattttag ttacatgaag atacttttag tagtcactgt tgttgttaca 58740 ttccccctac ccctcagaaa ataacagggc tgttaaggaa tcctcaaaga gcatagattg 58800 agtgaaaatg acacagaaca aaggacagat cccaggagtg aaatgaagaa gttctgtaaa 58860 tagctgtgcc gttttccttt gaaatgtctg gcacactcta tatagtatag tggtcgagga 58920 tagaggggct aattctgtga tgaagcattc tgcatggaat ttcagcaggg catagatggt 58980 gagggcatct ggatctgttt gagtttttaa actaattata tgacaaactt tttaaaatgt 59040 agcatacctg gaaacatcat cccgccacgt catctgcatt taaataatca ttgcctcacc 59100 tttaacaccc agcctctaga aaaataaagc atgaacaaat gttagctctg agctgctctc 59160 agtaatttgg agtgtgctaa gcaaaaccta cattttctag catgaaattg gcacaggaat 59220 gcatgcaaag ttttgcttct ttgattcatc tggttaccta gaacctgagg ccattctgta 59280 ttctgtggac aaaaccctat atacctttca tgacccagct taaatgaaaa gtctaatgca 59340 aaacatgctg aatttctgtg gccatattta attgttctag tggtcccaca ggtatagttt 59400 attattcttt ttaattcccc tttaatggca atttctcttt tcctttttta aaaatgtctt 59460 ttgccttctg ttcatttatt acctgtctcc cttttttatt gtgagcatgt ctcttgcaca 59520 gcacagacac atagtgattt cttgttacat gtgggcccac atttattttt tatttctatt 59580 tgtttttcat tcctatttat ttatttattt atttatttat ttattttgag ccagggtctt 59640 gcttcattgt ccaggccaga gtgcagtggt gcaatcacat ctcactgcaa cctctacttc 59700 ccgggctcaa gcaatcctcc cgcctcagcc tcccaagtag ctggaactac aggcacacac 59760 caccaaccac acctggctaa tgggcccata tgtaaatgac taattagtat ttaaacactt 59820 gccaagagtt gtttaatacc tgctgtgtgc agggcacaat cttatacatt cttaagggag 59880 cacagaggtg tcagatgggg tctgcccacc agtttttcac agatttgaat atttagcgtt 59940 ctgggcaaaa gcagtttagg agctgaaaaa tcatggccat tttaagaaga ttatcttacc 60000 caaaaaccca cagtactatt gttattgcct tatatataaa gccattctgt taatttttta 60060 aaagtatgta ttaggtttaa cctggtttct ctgtgtttgc catgtttaac atctaaggcc 60120 aaaaatttga aatattttta ctttctgagt atgtcaggaa agaggagtct gaagctggag 60180 tgagttgggg aggtgggggg gactatattt ttatgaaaat tttagtaaat ttcctgggtt 60240 tttgtcccac ctatggggaa gatctaggat acaaaagcat taaatgttaa tattgattca 60300 agaattaaat catcaaaaca ttgctgggag aaattaaaga cctaaataaa tgtagagaga 60360 tcatgtttat gggctagaaa actcaatatt gttaagatgt caccaaatta atatatagat 60420 tcaatgcaat ctctatcgga atttgctgaa attgaccaac tgactctgaa attcatgtgg 60480 aaataaaaag gatctataac agtcaaaaca accttgaaaa tgaaggataa agttggagga 60540 gtaatactat ctgattataa gacttattat aaagctatgg taaatcagga tattgtggca 60600 ttggtattaa gatcactgaa gagaccaatg gaccagatta gagcatccag aaataaaccc 60660 atacatatat gcatagctga tttttgaaaa agttgtaatg gcggctcagt tgttccagga 60720 caattagata tagttggaac aattagttat ccatatacaa aaaaagaaaa gaacttcaat 60780 cagttcctca taccatatat aaaaattaac tgaaaatgga tcatagtcta aatatattac 60840 ctaaaactat aatacttgta gaaaggagga gaaaaccctt gtaaccttga attaggcaaa 60900 tatatatata tatatttttt tttttttcaa gcagttctca tgcctcaccc tcctgaatag 60960 ctgttgttac aggtgttccc caccacacct ggctaatttt tgtattttta gtagagacgg 61020 ggtttcacca tgttggccag gctggtctcg aactcctgac ctcaagtgat ccgcccacct 61080 cgacctccca aagtgtgctg gaattacagg catgagccac cgtgcagagc cacaaaaata 61140 ttttaagtag aacatcaaaa gcatgatcca taaaataaca aatagatgaa ttggacttca 61200 ttaagattaa aacttctgtt tttcaaaaga cactgttata agaatgaaaa gatgagccac 61260 ggagtgggag aaaatattta tacatcatat aaaagatttg tgtgcagaat atataaataa 61320 ctctcaaaag tcaatgtaag aaaacctttt tttttttgaa tgtgcgaaag atttgaactg 61380 actttttgcc aaggaagaga tacagatagc agtaagcata ttagtcatta aaataatgca 61440 aattaaaact ataatgggat accactatta gtagtatgta ttagaattac tgaaattgaa 61500 gatcgacaat accaagtgtt ggtgaggatg tggaggaact agacgtctct tacactgctg 61560 atgagaatgt aaaatggcag aagcactttg gaaaataatc tgacagtttc tgaaaaagtt 61620 aaatgtatac ctagtgcatg atccagccat tccacttcta tgtatttatc ttaaaaaaaa 61680 gagaaagaaa ttcgcataaa cttgtaaaca cgtatttagt ttgtagcagc ttcatctgta 61740 atagccaaaa gcttgaaaca acccaaatgt ccctcagcag gtgaatgtgt aaactgtggt 61800 atgaactacg caatagaata ccatccagta aaataaagga gggaactatt aatgtacatt 61860 acaagtagat gaatctcttt ataactatgc caaatgaaag aagctatatt tacacataca 61920 cacattcaca tacaccccac atgctgaatt attacattca tacaaaattc tagaggatgc 61980 aaactaatgt atagttatag aaaggagatt ggggtttcct ggcgatgggt gatataaaaa 62040 gggattggag aaatggatta aaagcacaag gaaatttttg ggggtgctgg gtgtgttaat 62100 tatattcatt gtggtgatgg cttcatggct atatacatat gtcaaaatgt gtctaatttt 62160 agactttaaa aagcgcattt tattctatat tagtatttca gggatcccaa gacaaccctg 62220 tttggtggtt tactaggacc tgtagcactc agcatatagt cgttcttagg gctaagactt 62280 attacactga cgtagaagga tacacaggtg gatcagtaag gggaaatgac atcaggcagg 62340 gtccagagga atccatgtgt gggttttctg tgttccctcc ctccctctga gatcacacac 62400 agcataccac ctctccagta gtgaaaatgt agtcacaggc gtacaatgat tatacccagg 62460 gaaacccact taagattcaa gagttcaggg tttttgttca gggcttgtca cataggcact 62520 cttgcccacc aatatttcat attcctgaaa ggaaagcagt tgttcagtgc cccagacaga 62580 taaaataacc ttataagggt aggaaatgtt tcaaaagctg tgttcccgga cactagccaa 62640 gggcaagcct tgcaagcaga cctttctaaa gataaggctt tagataaagt ctcaggcttg 62700 ttaacacttt tctgcagtca cttatacctc agcttgttaa aaaaaaaaaa agttgtaaaa 62760 gttagtgtga gttagcacaa gacagttgca gttcatttat tgaaaaaatg ataagagatt 62820 ataaaaaata gtaactttag atttgaggca gaccaatgca gtgagaacct cattacctgg 62880 aatgcttcat acccttaaat cccctctttg gaattagcca ggagtgttgg tgggtggctg 62940 taatcgagct actcaggagg ctgaggcagg agaattactt gaacccggga ggcagagatt 63000 gcagtgagcc aagatcacac cactgcactc cagcctgggc aacagagtga gactctgact 63060 ccaaaaaaaa aaaaaaaaaa aaaaaaaaat cccctttttg gaacaaggta ggataataat 63120 aagtaaaacc aggaagcttg gctaatggga gcttctaatt atttttactc tttaatattg 63180 tgactttcat ttctattgta tttgaaaatc tttccaaggt atatcatttt tttcctttca 63240 taaaaaattc cttggttgat gttcattatt taaccactgg ttaactgatt atgacccagt 63300 tcagttctat agcaaataaa agataaagca ctgctgttga aattataatt acctcaacaa 63360 caagaccacc tcatgcagtt gtgcaggtta tacctcgtcc aaggcacacc cagctgtgag 63420 ggtgcgtgga gcttaagtcc agcttgtact ttgccaagct atgggctcca gcatggggtg 63480 tgtctgcccg agaaaggggc acctttttct agtttatgct aagatgcctt ttgagctgtt 63540 cagcatgagc cctggaaaaa ggtggtggta gcgattttct cctacagtgc tgcctaatat 63600 cagcaagctt tcttgtgggc atctctttcc ccttaaagaa atccagctag taactttcag 63660 ttgagcgtca tgtttaacca ggattccact gtgtgtaaca aggacaagcg attccaacgg 63720 tctctagttt ggcccaattg cccttaaaag gcaggcaaat gtttagggta tgtgtttctt 63780 tttatcactc tggaagaagc caggttcagg tctgtatctg gatcccatta ttaatgtccc 63840 cagccaagaa ttctgtgtat gtatagcaat agaaaagtca gagaaaggac gctcctgtga 63900 cttctctgaa gtttttgttc ttcctgagta atgaagtttc ctaaagggga attaaacttt 63960 ctcacctcgg aagatagaag tattgtttaa agattcaact ttgaaacaaa taggaaaata 64020 tctgatagtt gcaacttagc ttcaaatcag cgatgtccag atattgacaa ccattttatt 64080 tctcctaaac cttgagccct agcccaattt atgaccgagt agagatcatg gcacagcagc 64140 cagcctaaca gatgggggcc atactctagc agcagaagga ttctgttttg aaaccttcag 64200 cttttgaaat cttacaccac taccagccct acttcttcta catagatcac attcatttcc 64260 tccctctttt tcctaaggaa acttaaaaca catgtctttt ccctttttat agatgcgaac 64320 cacccgcaag gtctccgtct ggcctgtggg cctggttggt gggcggcgct acgaacgtcc 64380 gctggtggaa aacggcaaag ttgttggctg gtacaccggc tggagagcag acaggccttt 64440 tgccatcgac atggcaggtg agcagtgtgg gtgatgacat ttgttcctgc tggactgttc 64500 ttatgctctt gtttataacg atgcactaga tgaaggaagc tttaaaagac aaatttggat 64560 aaaaccctac atcatatcat attcaggaga tgaaatattt aaaccttcaa aataggctct 64620 ttggttagat caagtggaaa ttgatttgga gggtgattag aaaatgccct ttctgttcca 64680 ttttgtcccc tgtctgtgcc tttatcccac aagcgtctac atggcagcta ctgggttggc 64740 cactggggat tcggtggtga gagctagacc tggctcctgc cttccagggg tctgttagct 64800 ggaacctttt gactcccttt tctagaagac tgatatatta aaatatatca tgtttattga 64860 taaatttaac aatgatataa aaattgacaa gtcattttaa tgcttattga taggtttagt 64920 gctttattga tatatttaat actaaaatat aaacttagat ttctggtatt tcaagaattt 64980 attgagtata actgtcttaa agtttttatt ccctgaacaa ccaactgaat tgaattagcc 65040 ctgcagtagg accacacgtt ctaaaatgct actttatcat aaaacaactc attttctatg 65100 ggttatagaa aatgatgcct gtgataactg tggaaggatg gagtttgaaa catgaggctc 65160 agaggcaaaa aaaaaaaaaa ttcggtgaca tctatactct taaaaatgaa ggcagaaagg 65220 cttcaaaatt gatctcatca atcttgagct ctgaagcaca gctctgtcgg gctgccagcc 65280 tgtgcttgcc ccatcttctg tgcatatttc ccagccgtag tcaagatctc cgcgttcaga 65340 attctgatgg gatggaggct gtcttctgaa ctggattcat cacttttatt ctgacaatac 65400 tataaatctg agggagccag aggacgaaga ataccaggag tcatctgagg ggcacagcgc 65460 tcatggacag ctgtgtggtg ggtcttgtca catggcatcg tgggagagga caaataggag 65520 atttttactc aacatttaac acacatctcc acagcaccag ccctcagcca ggccatgtct 65580 caggcccctg acctctccat ccctctgtta ctccagctgt ttgctttggt cttcagttct 65640 tgcagctcca gaactgagct ggcatctcag tctaggtctc cacctggagg cagaaggaga 65700 ggcagctgag ccatcttgga cggtcctgag gaccctcgaa agaggcctgc cctcgacccg 65760 ctcagagtgc atggccctga aaacatgggg catagtgtat cttttgaaaa tcgggcttct 65820 tgctgaccag aagtgtacct tagagcagaa aatttagaca acattaggga aagtccttct 65880 gcttctctgg gatgtagttt tcttgtgtaa agtgaggcag ctgccggaga tgaccaccag 65940 ggccctccag tgcagccctc ctttcctggc ctcccacctt ggaactctca agtaagagcc 66000 caagcttggg cttctaagtg aggattgtga ctgctcaagc agccgcagac agacttttgc 66060 ttcagatttt cccagagcct tgtttcaaca cggacttccc agacatatct ccttaacacg 66120 tgggatgtct gcccatttcc ttgcatggat catgcaaggt gagggcaact tgggtaactc 66180 ttcctggatt ttaatctagg aactttatgt agatgatcac gtatcaaata gacttgttga 66240 atatttgcat tggttcccct agaatttggg tcacattgat atttctaaaa ttaaagatgt 66300 atgtgtttgt gcattagggc tggcctatct aggactgcca aattctgtgg gaattctttt 66360 ttccatgaca agaccaatgt ttggattttt ctaagcattt cagtatattt tcaaagcata 66420 ttaaaaacac ccatcataat aagatatgaa ttatttcctt gctctgtatt cattatcatg 66480 gttttaaaat aggaataaag tgtctctaag gagacaactt ttgtcacact aaataatttt 66540 tttcacttca cacatgaact tgctttgcat cttagctttc agtaaattat tgatattttt 66600 ggaagtcttt aagtgtaagt aatttggtcg ataggatagt tattctgttt ttgaggcagt 66660 ttgttctaat tctaacttaa aaatttgtgg ctttgtttag attccacata caattcattt 66720 attctctggg aagagggaaa agacctttta ggccttcatg ttcaattggt gcttcccgcc 66780 tcatttccaa gttacatgta cacacacaca cacacacaca cacacacaca cacacaatct 66840 gagctctgag gagctctact ttgaaaatgc tctgtgatag tcaagtgaga gcaccatatt 66900 ggagcctggg tctgtcttgc atttgcaaag cggattaaga gcccaaatgg aacatttgtg 66960 ggtttgagca cattcagtag aagcccctca gagcctgtcc tgtttgtatc tctgtgaggt 67020 catcacatag cccatcactg gaaactcatt tgagtatcaa acattgaaac tgcgatgttt 67080 catgtcattc tagtagttct tacactgtca ttttattgcc atgttgaact tgcctgatac 67140 tgaaattttg ctctcaggtg acatgaaaca ttgttcttga aataatctta tgtttacaac 67200 cataatggca gaattaagat tatttctgtt ccattggagg atatttttta aaataatgat 67260 tactacttaa aaaaattaat atgcctgccc acaagatgga aagaaaacac tggaggcagc 67320 cccactggtg gtgtgcagtt ggcttcaaag ctgaccagta gccacatagc tctgccaagc 67380 cagtgaggcc ctggggggtt tctactgttc tgtgcctcag aattcaattc ttacacctca 67440 ggaatctgag gattggctaa aagtgaggaa gtccaccaaa ggttttagct ctgttttgaa 67500 gggaggcagt taatttcaag tgacagactc acttcagcca cttccaggga ggtgtcagta 67560 agaacgactg cttgaaagaa ctgagcttaa atcaatcttc accctcacca ggaggtgtag 67620 atttgttcaa gttgagaact taaaacagat atttattagg aattttcaac attggcatgt 67680 gccttattat taattctagg atgtggcccg tgggacacgg tgctccatta atgacagtat 67740 atgcagttcc cagttgagtt gctggcacac agtaggtact ttattctgct aaaaacacaa 67800 ctaggtgtac tgcaattaga ttctgacacc acctggagtt ggtgcagacc ccacaggtta 67860 gagggcacag tctccaacaa gactgccctt atttcacatg ccccccacgc gctggagagt 67920 cccaggccac ctgcacttct gatcaactga ctgcactgga tgtttcccac aatctcctca 67980 ggtttgctaa tttgctagaa tgactcacag aactcaggag agtgctgtaa ttatacaaat 68040 tatcattgta atcagtgtta taaagaatac aagtcaggac cagccaaatg aagtgacaca 68100 caggccagca gagccaggtg gaaggagcag gctgggagca tacaggtctt ctgtgccttt 68160 tcctcataca ggtgaagtct aggaaagctt agttttctgg tctggagtcc tgtggaccag 68220 ggtgtgtcga agccctgggg atggatgtgc tcccacaggg agactctgtg gcagcctcgg 68280 attgcatcct gggggcaggc atttaaagtg actgcagggg gggagccagg gaaagagaca 68340 aggcccagca agagggtgtt agggtggaca gtggcgaggt cactggagag aaggggtgga 68400 tgaatgatga attccgttat tggatgtggg aagaggagga cccttggtga gtatgggaga 68460 aagaccgctg gaagcagaat ggtctgagca aagttaggtt tctggacggg catggtagct 68520 aacacctata atctcggcac tttgggaggc tgaggcaggt gaatcacctg aggtcaggag 68580 ttcaagacca gcctggccaa catggtgaaa ccccgtctct actacaaata caaaaattag 68640 ctgggcgtgg tggcaaatgc ctataatccc agctactcag gaggctgaga catgagaatc 68700 acttgaacca ggaggtggag gttgcagtga gccgagattg cacctctgca ctccagcctg 68760 ggagacacag ggagactgtt tcaaaaaaaa aaaaaaaatt aggtttctaa tggagcagag 68820 ggggagagat ttgatctggg aaaggcttgg ggaggttctg tcttggctgg acagggattt 68880 gaaaggatgt tgtgatgatg agtggccagg aggtatgggt taggaagagg aagggtgcaa 68940 tggccactga ggtcccctcc aactcgaggg ttgtgtgatt ttgacaagtt ctgattctaa 69000 ggactgaggt tgtgtaaggc ggtgcacctg gttatacttg tcttctgtcg gcagccaact 69060 cctcctcgcc tacggcctat caccagcttt ccagctggtg gtagttttat tcaatggagg 69120 cttaaaagcc ctgacttttg tggcgagtgg gtggacagtg ggggtgggga gcatgactag 69180 gaggcaggag ggccaggaga caactcttaa gaaatgcggc agcaaatgag taggaaagac 69240 agatggtggc agtttcaagg ggtataacag ctttttcctc tttctttaat gatggagggg 69300 gagaaattga agttttaaga agtgataagt gttgaaagtc ccaaaggaga ggaaatgagg 69360 ggagatcaag accttagatg gacaggttca cctagataga gtcacctctg actcccagaa 69420 aaaaggtgag ataagtaagg aagccaaggg ctgggaaggt ggaggggcca gttgttggaa 69480 ttgaccgtcc gccaaatgtt aatagaggga gaagtgacat ttggctgaat tccacacaca 69540 ctgaatgtag cagcccctcg cctcaaggag ttttgtatct agcagagaga gaatgtctac 69600 aagagcatgg cagatacatc gtggggtcga aaatgagtac aagtggaatg attgggggtt 69660 acatttaaag agaaaaccac agccactggg ggagtttcgg gaaaggtctt gggaagaggt 69720 gacatctgag aggcaccttc cgtgctaggc ccaggccagg gtagggaggg atcatgggca 69780 gggtgtgttt ttggaacaag ttatcccagg gtatgtgtag ctcttgggaa gggcaagctg 69840 gggtcagata gtgaggactg agtgctaaga tgagaagttt ttcttcattt ctgtggacat 69900 tggagagtct gacatttttc aagtagggga ctgatgtgat tagaaataca gtttgagaac 69960 cacatgttgg cagtgtggga tgaatggggg aggagggctg ggcctggagg ctcaggcagc 70020 catctgggta atgagccggt agcgggctgg gaggtgagag gaggtcttaa ataccacagc 70080 tattggggac ttataaaatt ttaacagcct tttcagatta tactctaaat cagcagacac 70140 ttagatcttt ttaacacctt tttctgttgt atggtatggt gataaatgat gtattaaatt 70200 taagtttggt taattttaat tggtgtaatt tgtattatcc tttgagtcgt aatttatgtg 70260 tagaaaatac agtgatgttg ggtaattaaa acattctttt aaccagaaca cctattttgt 70320 gtaaaatagg agtctgcata ttaggaaaaa aaaaaaaaat cacatcacct gactgaagtt 70380 ttcaagctca ggattcactg tgggtgtgtt gtcccagtgg agggtgtcga ggctgaaagt 70440 gaggaagaca gtgatcacgc ctggctgggc ctcctgtatc catacctgcc ctctcccgtg 70500 tccactccac ctcgaagcag gaggaatctt tcaggaatgc aaaccagatt cttggcagtc 70560 ccttgctcat agctcttcag gggctactca tgctcttgga ataaacaatt ccttgtcact 70620 ggcacctgcc aggccctgcc cggcccaccc ctgcccacct tactattctt gttcccctct 70680 actcaatctt tgcgggcaga gatcacttcc tccagcacat tctacccttc cctttggggc 70740 ctgcactgat gcccctgcct ggaaatgctc cttcctcgtt cagccctact catccttcaa 70800 atctcagctt aaaggctgcc ttcttgggga aggttttgct gatgcttcaa ttaagatagc 70860 tcctcccgtg ttataatgtg ctgttctctc agcctcccat acctctgcac cttgtcacag 70920 tggttgtaca tctctcatta taattgttga ggctccgcca ccccgactcc cagaatgcat 70980 ccttcctgta ggcagaagcc ggactatcct gccctccact ccttgcccaa tcccagcccc 71040 aggtttcccc tagccccagc ctgtgagtgg ggctgagtga cggcactatc ccagagcagc 71100 tgtccccgct acaagtttac caggcaaacc tttaaaaaat tattataaat gatgaccatg 71160 aaactggagg gggtcgaggg atcactctgg gcaggttgct gaagcctgct ttctgtgggc 71220 tctctgcagg gacatgggaa tgacagttat tctgggtctc cttcatctca atgtttgtca 71280 acaaggaatt ttgcctgggt taatttattt ggcagacctt ttctcagtag ataatgctgt 71340 gatcagcttc agcccagccc ggatcagatg atcatcaaag ccaaatgagc agtcaaaatt 71400 aatgacgttt tgctttgctt catgaatata aatactgcaa gaaaatggag ggaattgtct 71460 tcctgccact ttggagtcat tcgtgattta agtgtgctgt tttccatgca tgaatgtttt 71520 ctatgagaac tataaagtta ctgaatgttc tcagtagagt gacttgatgt gtcatgtggt 71580 accttttagt gcaggatcta gggaccagct tgggactttg tccttgggtt ggtacagtgt 71640 gattgtcacc gggagaggac tgcagctgcc agggggtggt aattctgtcc caacaactta 71700 tagaaccaca gggacaggtg gcagagtgtt ggggcaatag gcagcctgcc actcagtttt 71760 taatctattt ctagaacatg gtgcagtcta gagacttgca gggatttgat gcccacagta 71820 ctgtgtctgg tcctgtctgc atgtgctgtg gccagggctg tgctgggtag aggtgggcgt 71880 gtggggcaag gagcacatgt gcatctgtgt gctcatactc agggtgcttg ctctggagca 71940 gctggtagtg gggtcaggtg gggtggacgt ggagagggag ggctctgcag aggcctttca 72000 gggctgaagg ggaagtgggg agacgggttt ccaggcctct gtccacctca attctagcag 72060 ctctgcattt acatattgga gatcccctca agatttcaat ggaataaaac attcattcca 72120 ggactaaaaa ttttgaaaac ctgaagtttt cctttctatc aggatgtcca gcagactcaa 72180 taattatata ttgtttgctt agcatttacc aagcatcagg catttggaag cattctgtgt 72240 tctttcttca tgaatcctca cttagctgtg caagggatat gctagtttta ttatccccac 72300 ttacatataa ggagcccgag gcctaggtat gctaagtgag ttgtctggga ttcagagtca 72360 ggccagtgtg tctacagaga tggtctccaa ctcacccacc tacagccaag tcactgagtt 72420 cttggtctct gagcctgcag aacagtttct tggtttcttc atctgcagaa cagggaaaat 72480 gaaatttttc acaaaatcag acatcacgtg caaagcagcc aggagtagaa cattgtagac 72540 acttggtgaa tgtcactctt aaccaagaaa caagactgtg cctttgggtt cagctggctc 72600 acacatttat tttgatgaat taggtcagtg ttttgtttga ttatcacagt ggtgaggtcc 72660 atgcaggtag ctgtagggtg gaagaatcac tcatccttgg gtcctgctct gacacctaca 72720 ggctgtgcag cctgaagatc taggggaata tctgtttctt cctaatacct gtagattagg 72780 gatcattgct tcttcctaat acctgtagat tagggatcat tgcaccagct ttctgggttg 72840 tgttgaggtt gaagtaaaac gaatacatac agaagttcct gctgttctgt gaatatttga 72900 atctgcaccc acttgtagct ttgtgagatc actcttaacg gtagtattta agaacatttg 72960 aactccgctg tgggctcatg atgaacttca tttctcttct ggcgggtgga cctgtgctca 73020 ttatcattca atgaattggc tcagcatgca ggatggcatg ctgagataaa cgctagccct 73080 tactttagat taaatacccc caaaagagag tgatcaacag gagaaaatcg aagccaaaaa 73140 agatcattaa agagttgttt aggagcagat acgtgttcat tgttaaaatt tcccagctga 73200 aaatctaaac aaacagcact tgagctttca gaagaaaatg ccatttgtaa cattgagatt 73260 tgcaaggcat ttggtgccat gtgagtgccg cttgcccctg taggtgagtc tatgtagacc 73320 acgagacaag tattcaatat gcaaattcct gatggcacat agggaaggat ttaaaagaat 73380 gattctcatg ctttctaatc aagtcacaag ggggcaaaat gtcctttttc accacctcga 73440 ctttcctaag agtccctagg agagcatctg taggtaatag ttttcatcta gaatctgtta 73500 aataggtatt atttatttta ctcatataaa tagttaggat taagggttgt tgccctgcac 73560 tgctaacgtg ggggccgggg tgcagaccac ttctctcttt ggcatgcagg ttgtttgctg 73620 tgactactac acatttttct gcactggctt cagaagttaa gccttgcagg cattcttcct 73680 gggctccctg agcagctgta atcaccctga ccccaaacac tctgcacatc agttcacctc 73740 aattttaaat ccctcaggcc aaccgctgtg gtcagtcggc attgccctaa gctgatgggc 73800 ctgattaaac caagagtggc acacactgcc ctccagccta ggatatgcaa tgtcactttt 73860 cttctcacgt ttgcctagct aatttccagt tactcttcca gactcattga tgaggcagga 73920 atcgcttttt ctgggaagtt tttcctgatt ttccatcaca cgtggatgcc cctttctacc 73980 tttcccctag aatgccttgg cttatgcagt tatgaatatt tccacaaggt ggcgctcatg 74040 tcagtgtcct ccctgcccct ggccgccttg ctagccccgg agtacactag gagggcgctc 74100 gggtctgcct gcatctccag gccgggcaca gtctctgatg gtaatagctt gaacaaatgt 74160 ttgctgaatt catgaatttt aacatgtttt ccttttctag tgtaaggatt atcaaaatta 74220 agatttaaag cttctcgctt ctgagacaac attgccatta tttaatggtg gaggtgtaaa 74280 gggggatgcc gagctgtata ctccagagct ctctgaggag ggtctaattc agcgggtctc 74340 cccatggctt cgcatcaaaa tggcaagggg ctttgaaaaa acacccacac ttgagctcca 74400 cctaggagca aagagaccat atagtctctg agggtgaggc ctggtatttg tgtggttgcg 74460 ggtttttttt gttttttttt ttttttttga gtcttgaggt gatcccgaag tgcacccagg 74520 agtaagagag gcgggcgaaa gtgatcagaa gcgactccta tttccttcct gtggctcgtt 74580 atcgtctctc agtggtcaca gtcctttccc aggccacccc ctgtgtccat ctcaggtggg 74640 ataacctcat gacagagttt gggaacgctc aatagagtca cttccataga aaatacgtct 74700 tccattgctc aagcaagaag agaatttgag cataggacag gaatatttta atcataataa 74760 ctgaaaaaca ccagcaacaa tataaacaaa aagattgcct gtattcattg ggaatgcaag 74820 ttttatctgt gttttgattg tggtgaaata tacgtaacat aaaatttacc attttaacca 74880 ttttgaagtg gacagttcag tggtattcaa ttcattcaca ttgttgtgca accataacca 74940 ctgtccaact ccacaccttt tccatcactg aaccaaaatt ctatgctcac taaacaataa 75000 ctcctgactt gcccctcccc tcagcccctg gttaccacaa ttctactttc tgtctcgatt 75060 aattagacta ttctaggtac ctcatataag tggaatcata tttgtccctt tttttttttt 75120 tttgcttatt gagcataata tctttaaggt tcattcatat tatattatgt atcagaattt 75180 cattcctttt taaggctact aatattccat tgttacgtat agtccacatt tcgtttatcc 75240 atgcatacag ccatggacgt ttgggttgtt tccacctttt ggctactgtg aataatgttg 75300 ctataaacat tggtgcacag atatctgttc gagtccctgc tttagattct tttggatgtc 75360 caaagtagaa ctgttggatc acaggtaatt ttttgtttga attttttgaa gaatcaccat 75420 actattttct acagcagctg catcatttta cactctccac agcaatgcag gaaggttcca 75480 gattctccat atcctcacta atacttattt gcttctgttt tgttgtatta gtttttttaa 75540 taatagccac ctaatgggga tgaagtggta tctcattctg gttttgattt gcacttcatg 75600 tgcttcgtgg ccatttgtac ttccttctta gagaaatgtc tattcaagtc ctttatttct 75660 ataaaatgtt tatgtattta tttgacttca ccaacagaaa tggattttct cacagttctg 75720 gaggctggag gtccaaggtc agggtgtcag catgtgagtt cccttgaggc ctctccttgc 75780 tcacagatgg cctttcctct gcggcatgca ttctccactc tctcctcttt ttatatcagt 75840 catattggac tttgcccatt tttgaattga gttgtttggt ttattcttgc tgagctgtag 75900 aaattcttta tattttgggt attaatcctt tatcagatat gctatttaca aatatttttc 75960 ccattctatg ggttaccttt cccactctgt taatactgtt ctttaatgca ctaatgcttt 76020 agttttggaa gaagtctagt ttatctattt gatttattta tttttatttt ttattttttg 76080 agatgtagtc tcgctctgtc ccccaggctg gagtgcagtg gcatgatctc ggctcactgc 76140 aagctctacc tcccgggttc acacttttct cctgcctcag cctcctgagt agctgggact 76200 acaggcaccc gccaccatgc ccagctaatt ttttgtattt ttagtagaga cggggtttca 76260 ccatgttagc cagtatggtc tggatctcct gacctcgtga tctgcccgcc ttaacctccc 76320 aaagtgctgg gattacaggt gtgagccacc acacccagcg tacctatttg atcttttgtc 76380 gtctgtgctt ttggtgtcat atctaagaag tcactgctga atccaatctc atgaagcttt 76440 cctcgttgtc ttccaagagt tttatagttt gagttcttta gctttagtct gctgttagag 76500 aaagaataac aaaccttcat ttatccagaa tggttgaggg ttgggggcag agtatggtct 76560 tttttataat taactgtaat aaattttacc atttctacat tcctccatcc attaaaaaga 76620 tcaggattag aaagtaggaa aatatctaaa accatgctgg aaaacagaaa aggacatgta 76680 caatgaagct tctgggagaa atgtgaggct aagtttggat ggacagacag acagaggagg 76740 gggatcatat gagaagtaag aggatggtgt cctcatggga gcccctggac acccctattc 76800 tcagagtgca gagatccagt gggctgaagg gggccgtgcc tcagacacat gccatcaagc 76860 ccctacacag cttcctcgca tggcagagaa tgcccccaca tcaggtgggg gttgtgtggt 76920 aagctcctgg ttctgcctga cttctgggca gtttcctccc attaacatgg agggaaaggc 76980 tctatcacag cccactggtg aagccgcctc tgaagtgttg agtttccctg agtaactccc 77040 tggtttttct gtggaaaccg ctcaaagtgg ccacaggtgt acctaaaata aagcctctca 77100 caattgctaa caaagaccaa tacaagccat caatgctgtt cagccttgac ttctgtttgc 77160 aaatatgagt agataattaa aaaaagtaaa ccaagacaca ggagttccca ctgaaagagt 77220 ataggaatca agagagggct tttaaaattt ctagagttca ttctcagatt ttaaaagatg 77280 tggcatcata aaacaagagc cagcagctgt gaggaaagag tcattggaga gcaaggattc 77340 ctgtaaataa aaggcatgac tgctggaata acctcatgtg agagggtgac agcatcgagg 77400 ctctaagtgt cagtgatact gttagggttg caatgcagag gagtggatcc caaagccctc 77460 ctggaagaat cctccaggaa ttcttccaaa ataggagcaa aagtacaaag aaatagaaat 77520 gtcagtggaa ggagacttat aaatccagca tgttcagtaa gagtttcaga attagaaaat 77580 aactatatat gaaatatcaa gaaagaaaca gggctggttg tggtggctca catctgtaat 77640 cccagcactt tggaaggcga aggcaggaga atcgtttgag gccaggagtt tgagaccacc 77700 agcctggcca acatggtaaa accccatctc tactaaaaat ataaaaattg gctgggcatg 77760 gtggtgcacg cctgtgatcc cagttactcg ggaggctgag gcaggaggat tgtttgaacc 77820 cgggaggcag aggttacagt gagctgagat catcccactg cactccagcc tgggtgacag 77880 agcaagactc acagagcaag actctgtttc aaaaaaaaaa aaaaattagc cagctgtggt 77940 ggtgcacgct gtagtcctag ctacttggga ggctcagcca gaaggatcac ttgagcccag 78000 gggattcgag gctatagtca tgccactgca ctccagcctg ggtgacagag tgagactctg 78060 tctctaaaaa tcagaaagaa acaggataca agttcctata atacttgagt tttgagagtg 78120 attccattgc caggtttgaa tttgggctct gctacttcct cagagtaagc ttttaagcaa 78180 gttacttagc ctctctgtgc ctccatttct tcatttgtaa attatggtag taatagtatc 78240 tacctagtgg ggtcactgtg aggattaagc aggttaatac agacagagca cctaggacaa 78300 ggcctggttc actgtgaatg tttcctaaaa tgtagctgct gttaagccaa ccgtcactgc 78360 caccactact cttagttctt agaatgtaca ggtatgcggc tgccctgtga aagcttcaca 78420 gtcggtccgg tttaccactc cagtctctct ctctacctcc tctgccgcat tgccacctcc 78480 acccccaccc ccacccacac gccccggcat ctcttacctg gaccagcagt aacctcctaa 78540 ctagctccac agcttcattt cttatcccca ataatatgct ccctgacccg ttaggctcca 78600 ggttaaaaca tttctgcagc tccccttgca cttcgtttct ccatggcctc ccaggccctg 78660 catggttggg catccccacc cccgagcctc gctcaagtta ttttccttgt gttctctgtg 78720 cactcagctt cagtggcttg ctgtcccatc tcacaatccc gccatcacgt gtacttgtct 78780 ctgcctggaa ggctctttcc tcaacccacc tgcctctccc acaccatcct tcagagtcca 78840 gcttaatcat ccttggtcca ccctggaggg tcagatttct tcacatagca cccgcaccct 78900 tccttcatag cactcaccac cgtgtgtgca cctgcactat tagccaacat tccacctccc 78960 tgacaagcct gcacctgggc tataatgact ctaagatgca gatcatgact gattttgctt 79020 accaccttct ccccagtccc tggcagtttg cctggtatgt atgtttttgt tgaatgaatg 79080 aatgaatgaa tgaatgaatc ctaagtttag aacaactgtg ccaggattta ataataaaac 79140 aatcccaagg gtattcacag ggggtcaggg ggaaaactgc ttctctatga aggagggaaa 79200 aatcagacta gcatcaggtt tttttctgta acactgaatg atacagaata atggagaaag 79260 gtgtttagag ttctgaagaa aagaaatgga tcctaggatt ttttgtaccc cgtgaaatca 79320 ttatgtctgt gtgagggcaa aagagacatt tcaaatattt ttctcaaaaa acaaacaaca 79380 acaacaacaa caaaaacaaa gtcacttaag gaaagcgctt tagccaaagt gaattagaat 79440 aagaaactaa aaaataggaa gattgctgta ccagataaaa tagtaagcaa caaaagtggc 79500 aattttaaca attaaaagtg gatagtaact aacatgactg tgaagtttaa agctgtttgt 79560 tggaatctgg aaataggaga agcaaagtga aaatgaagtt ttcaaataaa aacgttggta 79620 tatttgaatg cctgctggtg acggagagga taggaaatgg gagaataggt gcccactagc 79680 agatgtagtc tcattcaaaa aggtaagggg gagggggggc agagagaggc tttaattcat 79740 gatgataaga gaaatatcag ttccttaaaa gctccttaaa cagttaaaaa tattttaaat 79800 aaggaatttt aaaggtaacc acagtagaat acatataaaa caaaaccttt tcaacaatta 79860 gaggaagaag agaaaccaaa atgaattcaa aaagaagaag gaaaaaaagc aaaaaaaaaa 79920 aagtatgtaa tgcacctaag ataaaaccaa acatgataac cacactaagt gtgaaaaaaa 79980 ttaaattgct ctaaataaaa cacagcagca aagctatatc tcatataaaa gacatgtaaa 80040 aatgtgatgg agatggacaa taaagagata aactatagta tcctaaggaa ggacagacaa 80100 caaaagagca ggagtgttca cattcgtgtt gaacaaggca gaagtcaagg acaaaaatat 80160 tgaatgagaa ggagaggaac attttgtatt gacggaaact atgtatacta caaaagtaca 80220 gcattcatca gccattgtgt atgagttatc aatgagtcca aaaactaaag gcagacatag 80280 ataaaaatag aaaatttgac aaaaccataa tcatagtaga catgtttaac acacttatct 80340 aaatctgaca gatttaagta gacaaaaggc tgtagaaaat ggaatatata aaaaacttga 80400 tctaataatt atatataaaa cactgtactc aatagacaca gaatatacct ttatttcaag 80460 tgttcatgaa acatttataa aaattgaaac tgtatcaggc cataaaacct taaaaaagtg 80520 cttgtatagg ctcaaagcac tacactagaa actaacaaat aataaattac ataataatga 80580 taattaaaaa gtggagttag atctgttgac aagtagatga ccagaggtaa actgacagca 80640 gggctggaga agcatggatg gtttgatcct tgctcagaag cctggtccct ggccagtctc 80700 tccctgtgct tccccatcag catttgcagg gaggaacaca ggattacaaa ggtcatggct 80760 ggcttatgga aacacaagct gaaattgaat ctcaggtatc tatgcctttg taccttagga 80820 cagtgtgttt tacccacagt tctttttaaa gtggggaagc gtggacagtc ttacaggcag 80880 gtgccttgag cagctccacc cccgccgcca ccacattcat gtcagtcttg gcctgaacat 80940 atcttcaggc tacaccaagg ttcaaagcca atgtaaggta agatttggct acaggatttt 81000 gcatgcagaa accatcagcg ctgttagaag cccctttgaa ggagtgagaa ggagaaaaac 81060 ctgtacagag gagagcgtac aggttactct aggtacttaa gaccaggact tgatggtaag 81120 agagcctaca ggaaatgtcc aggaagaacc catagctctg cagaagaggc agagggtcgg 81180 gggaaaactg cctggccatg gagtgagagt tggaccaccc caatcctgct ccttctcttt 81240 tgtgtcttgg gtgaaagagt gaaatgcctt ccaggttctg atgtaagctc cttctttgtc 81300 ccagtgtcca ggcctgcact ggtgaccttc tccagcactc tctgtacctg tgctgaacac 81360 ccccggtggg gcccatctgt ctgctcttac ctgaaccatc tagtgcagca gacactttgc 81420 tggtctctct acctcaggca gttaccatac tgctgccaga gaaattttcc taaaattgac 81480 ttttgattac aatctggagc caaggttgcc tgccacagag tagggtgcag agtcctgggc 81540 acaagcaact tgacagcctt gtcacccatc attgctgctc cagcctaaga cttggcctca 81600 caggtctccc ttcctctaca tggatgcttc aaagtccctc tcagatttca gtgtctccaa 81660 agcccttact cattctagtc agatagcatt tgcttctcca actcctgtgc tcccaaagct 81720 ctctacttac accttttata gcatctgtgg cacagctgtg cccatatctg ttcttctaat 81780 taaactagga gttgccagag ggcagagacc acattcagta ttgtgttacc aatactgaaa 81840 accagcaatg ggcccctaaa gagcttctga atgagtgaat aatatcaaaa ggaagatgaa 81900 aaatgaaaga taacatcctt tagattggtg aataagaatg ctctttttta atgattatgc 81960 ttaacaaaga atgcttacta tgtttccagg taaatatttg tttacatata ttcatttaag 82020 atttacaata accgtataag atgatagcta tctgcatttt acatttgtgt aaaccaaggc 82080 acagaacagt gaaagtacct tcctgagtca ctttaatagt agatggcaga gtaaggattt 82140 gaattcaagc cctccaagtt acaagccttt actcaactac cgtccttccc cgccacacta 82200 tttcattaaa ataggtcttt aggaaactct gaagatttcc agtcagaaac caacatagct 82260 tccatgtgaa actaggaggt gatgtaatag cttcattagt gaatccctga agagctacca 82320 tttaaaaaat tatttcacta ggattgggga ataagtacct tcttcaccat gctctgctat 82380 attttctggg ttggactaga gtggcttagt ttgaccaatt cagtagtacc atggcctctt 82440 acccaggcca tgtcaagcat gttgactctg ccatgcccag catatacaag taaactctga 82500 agggtttatt tcccatcaga gtctttttgg ttctaaactt cagtgttagt tgtgcccact 82560 aaaaaataat tcatggatat tttccgtatt cagttaatta aaccccttta tattatgttt 82620 ggttattagg atcagatttt ccatccttgc tcagcatcct gattgtttcc ttagaagatt 82680 cagttttcta tgatggtttt ttcagagtag agatttaaat gaattgttta tagattaaat 82740 aaagaacact tcttggaaat gcagagtgtt tcactttttt ccatggaaaa taatgactgc 82800 taaatacctt ttgaggaatt tttaaaatta gacttttact taagtttcac cttgaagcaa 82860 ttataaagtt tcagtcagta gtcagcagat taacagacct gtttaacaac caaggcatgc 82920 tttttcattc ttcttccaca ccagaagaaa ataattctaa attataattg gaagtaaatg 82980 cgggaagcta tggttctgtt tactcagtag gaaagggtag atttggaaga aaatctatca 83040 tggctgtgaa agaacacttc atgacatatg agcattcagt cttagagcca tgaaaattat 83100 aaaggaacac ttgcaggaaa cagtggcaat ttgggatagt aaataagcaa aggaaacatt 83160 cctttatata gccttctttt ctgctttcta tatttgattt aaatctagtg gaaatgattc 83220 ctaaatcaat tttactaaca catttgtttc tcacttggat agttttgaga tgccagggct 83280 ggaaagtaca gggccagggg aaaaaaaagt agttaagtag cagttttctg ttttcaatca 83340 tcaaacccta cattacagta ttttgttttt cttctatact tacctataat gtggtactgg 83400 gatttggggt tgcaaaaaga gtaatgtcat tgggaatgac agagtaagga cttctgaata 83460 ttctcatcca taaaaacagt gagaacactg gcaaaatttg tcaaagtcaa cttcttaaga 83520 actatggaaa ttagctaaaa gtatgcaaga atctggggag ttttgttttt tttttttaaa 83580 aaaaggctaa atcttggtaa gagcagtatt gtagtatatc aatttgccct atttccatcc 83640 ccttctctcc atttcctcca tggtagcttt gaaaaccaac agccctgtaa ttgtggtgaa 83700 aaccagcagc atagcagtta ctagaagggc agaacagggt tggaactcat tcagagctcc 83760 attttaagag aattgtcatt atgtgatctg tccagcaatt ccctagaaaa ccctgttcac 83820 aaggcttgtc tttatttgac ctcattcact agagcaaaca ggcttttccc tgggggcgtt 83880 tgtcaaaaac agcttgtggt gattgtttac catcacagct ccctgaggca ataacagttg 83940 tggcaaacaa gctaagcaaa aaacttcatt ccgtaggggc atttgaaaag ctctgacata 84000 tttctggaag gccttgtgga tgtgtaggac tatatgcatg cccagggcta tgtgcttgtt 84060 caagaaaatc cttacaaagt cctcagtctt ccccctctta gtactgtgag cccctgtgca 84120 atcaagaagt gaaaactaaa gcagttgtac attgcctggc tgagtgttga aagcatgccc 84180 aaatggacac agagcatctt ggcaaagact gagagactct ggttccaggc atttaaggaa 84240 atctgtgtcc aatatttagt tgacctctta agctaactga acacagactt cattgcccac 84300 actcagcaaa gaacacagac tttacaaagt tagttcaagg aagtcactaa aacgataatg 84360 acaacaataa aagaacaact aaccttagag catctgattt ccagagttgt cacattatat 84420 tatttcaaat gttcagtttt cagcaaaaga gtacaagaca tgcaaataaa caagaaagca 84480 taaccaatac acaaaaaaga aagcagttga tagaaactgt ccctgaaaat gtctggacat 84540 tatatttggt aggtaaaaac tttaaatcag atattttaaa tatatggagt ccactaaaga 84600 taaccatgtc taaatagcta aagaaaaatg ggagaatgat gtctcacaaa tagagatcaa 84660 taaagagata gaaattatat atttttaaaa aagaaccaaa tagaaatgct ggagttgaaa 84720 tgtacactaa taaaaacaaa aaattcaata gaggggctca aagcagtttc agcaggcaga 84780 agaattagtg aatttgaaac atagataaat tgatatgatc cagtttaagg aacagaaaaa 84840 agaatgacga aaaatgaaca ggacctcata gtcttattat tcacatatac taatatacat 84900 attatgagtc gtagaaaaag agagaaaaag ggacaaaaag aatatttgaa aaaaaatgtt 84960 agaacttccc aaatctgtat gaaaaacatt aaacatcgag gaagctcagt ggattccaag 85020 taggataaac tcagagattc atacctagac acatcataat cagttgaaaa ccaaacatga 85080 agagaatctt gagagtaaca aaagaaaact gaatatgatt aacaaaggct ctttaataag 85140 ccattgttta ttaacaaaga ctgatttctt atcagaaacc acagaagcta gaaggcagtg 85200 ggatgacaga ttcaaagtac tggaaggaaa aaaagaaaga tcaaccaaga attctgtatc 85260 cagcaaaact atccttcaaa aatgaaggag aaatgaaaat attcccaatt aacaaacacc 85320 atgaaaattc attgctagca catgtgccct acaagaaata ctcaaaggag tcctttaggc 85380 aaaagtgaaa ggatgctaga cgggaactgt aatccacatg aacaaataag aacactagta 85440 aaagtaacta cataggtaat tttgaaaggc agtataaatg taatctttat agctcttttc 85500 tcctgttttg aaaaacagct acataaacca gtcatcagaa atctggtttg aggagcacaa 85560 aatgtataaa gatataattt ttatgacaat aatagcccaa atgaggggca aagaatggat 85620 ctgtgtaggg gcaaagtttt tttatcctgt tgaaattaag ttggtattaa tctgaactaa 85680 actgttagaa attgaaatgt taattataat ccccatggca acctctaaga aaataactca 85740 aatgataaat gacaagagaa ttaaaatcat acactaggaa atatttgttt aataataaag 85800 aaggcagtaa tagagaaata gaggaataaa ataacattag acatgtagaa aacaaacttc 85860 aaaaagacaa atgtaaatca atttattggt aattacatta aacgtaactg gataaaaaac 85920 ccaatcaaaa ggcagatgga caggatgtat aaaaatgatc caactatatg ctgtctacaa 85980 gacaaacatg ttagattcaa agacacaaat aaattgaaag caagagaaga gaaaaaagta 86040 tatatcatgc taacaggaac caaaaaaaaa aaaaaaaaga acgggaatga ttatatgaac 86100 accagacaaa atagacttta agacgaactg ttactagaga ccaaaaaaaa gttataatga 86160 gaaaagggtc actctgtcaa gaagatataa ctataaacat atacgtgact tacagcagag 86220 ctccaaaata cacaaagtaa aaatgacaaa actgaaggaa aaaataattc atcaataatc 86280 attgcaacta tcaacatccc actttcaata ctagacagaa caactagata gatcaactgg 86340 gatagaagac ttgaaaaaca ctgtaaatca attagatgta acaaacatct atagaacact 86400 ccacccaaca agaatggaat acgctttctt ctcaaaggca caatggaaca ttctccaaga 86460 ttatatacca tcttaggcca taaagcagga cacaatttaa aataactaaa attacacaaa 86520 gtatggtctt ggtttatact ggaataaaat cagaaattaa taccagaaag gagtttgtga 86580 aattcacaaa tatatggaaa ttatacacac actcctaaaa aaaatcaatt tgttaagaaa 86640 tcacagggaa aattgggaaa tattttgaag agaatgaaaa tggaaaaaca acatatgaaa 86700 acctatgggg gtacagccag agtagtgctt agcaggacat ttatagcttc acatacctcc 86760 attaaaaaga agaaagatct caaatcaata acctaacctt ctgccataaa aaaacagaaa 86820 aagaagagta aactcaaaat aagcagaagg aagttattag attagagaaa aatacagaat 86880 ataaaaacaa tataagaaaa tccataaaaa tgttttttaa aaaccaacaa attgggaaac 86940 ctttacttag actgtcaaga aaaaggagag aagcctcaca ttactaaaat caggaatgaa 87000 agcacgaata ttaataccaa ccttacataa attttatgag aatactttaa aaaattgtat 87060 tgcaacaaat tagataacct atatgaaaat ggacaaatta ttggaatgac tattaaaact 87120 gtctcaaaaa aatctaaata ggcctataac agagagattg aattaataat caacaaaact 87180 tccctcagag aaaagcccat tttcagatgg ctttactggt gacttctacc aaacatttaa 87240 agaattacca attcttcaca aatcctccaa aaaatagaag ggaacacttt tgaaggaatt 87300 ctaccaagcc aatgttaccc tgataccaaa accaaagaca tcacgaagaa aaaaaaaaaa 87360 aaaaaaaaca gacagaccta tatccctttt gaatacagat gcaaaaatcc ttaaaaaaaa 87420 aatactagta aagtgaattg agcaacatat agaaaggatt atacaccatg agcaggtaag 87480 atttatccct ggaatataag attatttcaa atataaaagt aaatcaatgt aacacaccat 87540 atcaatacaa taaagaacaa aagctgcaca attatatcag tagatgcaga aaaagcacct 87600 gacaaaattc agcattcttt catgataaaa acactagaga gaagatcaat aaactaggaa 87660 taggagggaa cttcatttat ttgataaagc atatctatga aaaattcaca gctaacattt 87720 ttaatagttg aatgccgaaa gctttttccc taagatcaag aataagacaa gatgacccct 87780 ctacccattt gtatacacca ttgcactata gcttataaaa ggcatccaga ttgtaaaggc 87840 attagtaaaa ctatctccat ttatcaatga catgatcttt tacatagaaa aatcctaagg 87900 aacacacaca cataactgtt tttttgtttg tttgtttgtt tgtttttaag agattttctt 87960 gagggtttat ttacatggct gtttggacat ctctgttgaa aaggaaaact cttttttttt 88020 ctttttttta ttattattat actttaagtt ttagggtaca tgtgcacaac gtgcaggttt 88080 gttacatatg tatacatgtg ccatgctggt gtgctgcacc cattaactcg tcatttagca 88140 ttagggtata tctcctaaat gctatccctc ccccctcccc ccaccccaca acagtccctg 88200 gtgtgtgatg ttccccttcc tgtgtccatg tgttctcatc gttcaattcc cacctatgag 88260 tgagaacatg cggtgtttgg ttctttgtcc ttgcgatagt ttgctgagta atgatggttt 88320 ccagcttcat ccatgtccct acaaaggaca tgaactcatc atttttgatg gctgcatagt 88380 attccatggt gtatatgtgc cacattttct taatccagtc tatcgttgtt ggatatttgg 88440 gttggttcca agtctttgct attgtgaata gtgccgcaat aaacatacgt gtgcatgtgt 88500 ctttctagca gcatgattta taatcctttg ggtatatacc cagtaatggg atggctgggt 88560 caaatggtat ttctagttct agatccctga ggaatcgcca cactgacttc cacaatggtt 88620 ttactagttt acagtcccac caacagtgta aaagtgttcc tatttctcca catcctctcc 88680 agcacctgtt gtttcctgac tttttaatga tcaccattct aactggtgtg agatggtatc 88740 tcattgtggt tttgatttgc atttctctga tggccagtga tgaacacaca caactgttaa 88800 tgctaattaa atgagtttag caagactgca ttgatacaaa atcaatatat gaaaatcaat 88860 tgtatttata tacactagca atgaacaatc tgaaaattaa actaagaaaa tttcattcac 88920 aatagtgtca gaaataataa aatgcttagt aataaagtat gacttacaca ggaaactata 88980 aaatatcact gaaatgaaga actaaataag tggaaagata tgttcatgga ttagaagact 89040 taatattgtt atgggtcaga agacttaata ttgctaaaat ggcaatattc ccccaattaa 89100 tctacagatt caatgcaatc tttatcaaaa ttctggctgc cctttttgta aaaattgaca 89160 aggtatttgt ttctaaaatt gatatggaaa gtgatgaact cagaaagaag ttaggggatt 89220 tatacttccc aatttgaaaa cttataaaac tacaataatg ccatcaccag atggagcgac 89280 atgcacctta tagtcccagc tactcaggag gctgaggcag gagcatccct cgagcccagg 89340 agtttgagac cagcctggac aacataggaa gaccctgtct caacttaaag aaagcaaact 89400 acagtaatac agtgtggtac tgacataagg atagccatat agattaatga aatagaactg 89460 acagtgtaga aataaatcat ttatggtcag ttgatttttg ccaaaggttc taagacagtt 89520 caatgggaaa aaaagtcttc tcaaaaattg gttcccagag ttatgaatat gctaaaaccg 89580 cccttaatca taccatataa aaggacaagt tttaatatat gtgaattata tctcaataag 89640 gaggggtggt gggcaggaaa ggtggaaaca cacaacattc caaaaggcag tgtatgcttt 89700 gctgggaaga ggtatgaaag gagagttggc cggctcagaa ttagaggagg tcccacacat 89760 atctttcttt ctccagcaag ttccatgact gcactgtgtg tatatacttt ttaatatgaa 89820 agttgaaatc tctttttctt attgtttaac gtgggggggg aaagagaggc ttttgaaatt 89880 acctgatagc tattggtaac actaaacatg tgtcaagagc ccaatactct aggacaacta 89940 cccagagtga ttattacaca gaaaaagctg ttagtatctt tcttgtggcc aaactataaa 90000 agtcttgttg ccaaaagtca tcgtttagaa actggaacta gttcttattt gtaacagcct 90060 tgctgagatt agatgatgta tagttggaga agagagaatg tggcagtggg atgagcctat 90120 ggaggtgagg ctaaattgat acttacacaa tagaagaact gagaagcact attgatactg 90180 ttaactgtgg ctgaaggaag aagaggaggc ttgctgtact tcttcctttc caatatttct 90240 gccttttttg tgggggtggg agccttattt aaatggccag gatctccagg actttgttga 90300 ataaaagtca agagcagcca tccttgcctc attccaatct tcagggattt aacactcagt 90360 ctttcaccaa ttaagtataa tgttggcatt aggtattttt gtagatgccc ttattaagtt 90420 gagaaagttc acttctagtc ttagtttgct gagaggcttt tatttcttct tttcattatt 90480 actagatgtt agattttgtc aaagcatttt ctccagctat tgtgatagtc agattttttt 90540 ttttagtctg ctaatatggt gaattacatt gattgatttt gaaattttaa accaacattg 90600 caatctggag ataatcctca agtggtcatg atttattgtc tttttattgt tgaatttgat 90660 ttgctaaaat tttgagaatt tttctgtgat catgaggatt attagtctgt agttttcttg 90720 taatgttttt gtctggtttt ggtatcaggg caatgctggt cttacaggat aacttgagaa 90780 agatttcctc cttttctatt ttctagaaaa attcatgtag aattgctgtc atttcttcct 90840 taaatgcttg gtagaattta ccagtgaagc tctctggtcc tggagggttg tgtgtatgtg 90900 tgtggaagat tttaattata aacttaattt atttgataca gggctatttc acattatcta 90960 tttcttcatg agtgagcctt gtagcttgtc tttcaaggac tttttctatt ttgatctaag 91020 ttgtcaaatt tactatcata aaaagttgct tataatagcc ccatattatc attttaaggt 91080 ttgtagaacc tatctctttc atttctgagg ctggtaattt atatcttctc tttttcctga 91140 taagcatggg tagagtttat caattttatt aacttttttt tcctaaatga ctaggttttt 91200 atttcactta tcttctcaat tgttttctat ttctacttta gtcgtttctg ctctcatatc 91260 tattagtata gtcgttttct ccatttactt tgggcttcat ttgctttctt tttctaattt 91320 ataaaggtag gagcttaggt tattgaattc agacctttct tatccagtaa gtgcttaatg 91380 ttttctaaaa actagtttcc catctaaaca ctcctttaga tgtatcctac acattttgat 91440 atgtcttgtt ttcattttca ttcagttaac cactttatca tttccctttt gatttcttct 91500 ttggtttgta ttttgcttag aattatattg tttagtttcc aaatatttgg gatattttcc 91560 aggtatcttt gtatttactg atatctattt taattccact gtagtcagaa aacatatttt 91620 gtacaattta aattatttta aatgtattga gacagattta tggcccagac tatggtctaa 91680 ctatcgtgat aatgttccca tgtgtacttt aaaagattgt attctgctgt tgttgggtgg 91740 agtgttctat aaatgtcatt taggtgtagt tgaaaatgtt atttaagtct tacatatctt 91800 tcctgagttt tcaagtttgt aaatttttca gtcgtcattt cttcaaattt ttttccctgt 91860 ctccctcatt cattcacctt tgactctgtt cctgtgactc tgtacggtgt gtgtgtgttg 91920 gtcttgcctt ttctgtttca ttttggtagt ttctgtcatt atgtgttcga gtttactcaa 91980 cttttattct gcagtgtctc ctctgctgct aatcccatcc aatgtatttt tcatctcaga 92040 cattgtattt ttcatcactg aggtgtcttg ggcatcaggc attttgaatt ttatgttgct 92100 agatactgga tgagagagac atatatgtat acacacaacc atacataaaa acacatatat 92160 atgtaatgtt atactatatt ataaatacat atgtattctt gagctttgta ctgggacaca 92220 actgagttaa cttggaaaca ttttgatcct ttccaggctt gcttttgaag tttgttaggc 92280 aaaaccatct cagcctctag tttagggcta atcaatttga ccttacaaca ggcacgataa 92340 ccttccaagt tctcctccag gtgctccagc tgttactagg tagctccact ctggctactg 92400 agaatgggaa ctagtcccct ccagtaattg attcacctgc tccttgccag tggttctgta 92460 cctggcctga ggtagttgtg tgagcatttg ctcttcatac tcagttgaag agttgaggag 92520 agctgtctac agatcagcag agttcttcct tctctagtac tccaccctgc atatcctagt 92580 tgtcttggcc tcctcaaatt ctcagttgtt tcctcaattc aaggggactg cagagctttc 92640 cgggttcccc tccctgtggt gtagcctgaa aacatacttc aggcaggaag ctggggcagt 92700 tgtaggggtc acatttctct tttctcagag atctctgtct catgtgcctc ttttccaatg 92760 tctggaaact tttttcaatc attttgtctg ccttttcgtt atttaaaaca agtgaggaaa 92820 aaccagtccc tattgcaatt ttgtccagaa atgtaaatag cttgttggac ttttaaagtc 92880 ttacaaattt tctactcagt aatcagtaat catatatgga ggtaggccct atccctctgc 92940 ctgagaggga atagttattc ctgagtatcc atagggaatt ggtttcagga ctgctactca 93000 taccaaaatc catggacgct caagtccctg atatagtgtt tacatataac ctaggcagat 93060 cctcctgtat actttaaatc atctctagat tacttaccta atacaatgca aatgccatgc 93120 aaattgttgt tatactatat agctttaaaa cttgtattat tttgactggc tgctgtagct 93180 cacgcctgta atcccagcac tttaggaggc cgaggggggc agataacttg aagtcaggag 93240 ttcaagacca gcctggccga catagtgaaa ccccgtctct gctaaaaata caaaattagc 93300 caggcccacg tctgtagtcc cagctactcg ggaggctgag gcaggagaat cacttgaacc 93360 caggaggcag aggttgcagt gagccgagat tgtgccactg cactccagcc tgggcaacaa 93420 gagtgaaact ctgtccaaaa acaaaaaagc cctattattt cttgttgtat tgtcattttt 93480 ttgttttatt ttcagatatt tttaatctga ggttagttgg atctgcagat gcatctgcag 93540 atacagaggg tgactgtgta tcctccccac ctagctctgc tgaagaagtc aggaaatcct 93600 gccttctcag tgaacaatca tttttatttg catttttttt tttactcagc tgaccagggc 93660 agaaaatcct actcccatga ctttcgtaag catctccaaa gtaaccaaca tctttaaaga 93720 taacgaatct cactaaaaaa ggaaagatat aatacttgaa gtataaatac ttggaattac 93780 ccaagaaaac atcttaggtt ggaaatcaat ggtttaggat tccctttact gtgatttcag 93840 aggggagaac tctttaactc tgcccccagc ttcacagcgg gtcagattcc ccaaaagtaa 93900 cccaggggtt gacctagtta acatgcagtc tctcctgcca gctgtgacct gcttcctttc 93960 aagagatcac tgagcaaagg aaattgcgtt agctgattgt ggggcatctg aattgtgctc 94020 ctccacccct ttgtattaga gaatagaaaa acacttgggt actgtggaga gccacaggct 94080 aacatgtctc cagggtgctg gcctttcagg tgcgcaccat tctctagtga caccaggaaa 94140 ggagaccttg ctgcaaaatg atgtatagct tcacaactgg ctgttaactt tacataaatt 94200 gtagattttg ccatgtcatt ctgtgtgaag cgctggaagg atattcctgt gctaaggaaa 94260 gcaacaaaag aatctgaatc cttcagtggt cgtgatggtt aatgtgttaa agtcttaggg 94320 gaaaatgaag gaaatcagat ccaggtggct ccatatcatt tccagaaatc acctctgcac 94380 aatttggatc ccatgttttt gagagaatgg ggaaatacaa ccagtatcct gaagccatgt 94440 gaagaatgta tcaggagttt cagtatgact gagaagggta ggctctcatc tgaatttaaa 94500 aaaaaaaaaa aaaaaaaaag gataaagctt atttataagc atgtgggaaa aagcaagaaa 94560 tcttttaaca ctaaaatctg atctgacctg ttggaaagga cactacgtta tcaatttgaa 94620 cctctggctt ttgttgaggt gtctggtggt tgtcctagtt gtgtctaagg aaggctgtgg 94680 acttgttacc agagttgcta tcacatgtag gtgtcctggc tttgctacct ggaactttcc 94740 caaacctttt acatctgtca accagtattc tttcagccat aaatggtggc tggcatcacc 94800 tgcccctcat gatcacactg acaatagtat ttttatttat atattgcagt atcttttgaa 94860 cagtgctttg cagtcagtgg agtctttcca tacctcaatt tttcatcaaa cattgagttg 94920 aagtgttaca cttctctcgc aaataaagaa atggggaagt tagaaaatca agaaaggtta 94980 aatgagttgg ccagtgtccc aggggcaggg acctaggcaa aaacaaaagg cttctaattc 95040 caaatccagt attctctgct aaaatgtgcc acctccctcc ctttagggtt ggtggaggta 95100 tcgatactgg ggctagtcct acagctaatg ctttatgatt ctttgttctg cttcactcag 95160 cacctgctgt accatgttat gtttgtaagt ggcttagtgt taacttttct ccaaaaggca 95220 gcagggtctg gaacaggaga ctggcccagt ctggcatctg gagaggatgg tggttgtggt 95280 gttttcacag ctcctccatt accacctggt atcatttagt attactttgc aaactgaatc 95340 ataaatcaac tcatttaatg tagagaaggg caaaagttgc tgagaaatgt tttgtgggtt 95400 ggtgcccgga gcttcaactc tgggagggtg cctgacttga cagtatcacc ttagtcacta 95460 aggaaaaaga gatccagggc ttcagacctt caaaacaatt attacttgct gagatggcaa 95520 aaacaattgt agagtgcttc aatggctgag ctttaaacag tttgttaaac tacagattaa 95580 atacgaatga ttcttttttc tttgagacag ggcctcattc tgttgcccag gctggagttg 95640 cagtggcaca gtcatagctc acagcagcca caacctcata ggctcaggca atcctcccac 95700 ctcagcctcc taagtagctg ggactacagg cacgcaccac gtgccacaac acccagctaa 95760 tttttgtatt ttttgtagag acagggtctc attatgctgc ccaggctggt cttgaactcc 95820 tgggctcaag agatccttct gccttggcct cccaaagtgc tgagattaca gatgtgagac 95880 actgtgccca gccagtaatg atttttcgta tgaccaaacc attgaagaaa atttacactt 95940 atttttggta aatgaaatac caaaaaaaaa ctttatcaga tgatggcctg atatctcctg 96000 cactgatttc tgccccacat acccttttta gattgaacca aattagaaat ataaaccaga 96060 tgattaaaaa ttaggtatga ttcattttcc agctaatcgg ctttacttct gttgctccct 96120 cttctaaatg caaaatgaat ccaagcagtg ctggatttaa ggaagcatac ataattaata 96180 ttacagggca ggcaggagag ctgggcatgt ggtctcatac cccacatgta gttaggtaag 96240 catgtatgtt tttcagctgc atttatttct tagttattcc tagtggcaaa tgtggggggt 96300 tgatgatcac tgtgaaacag cagcttataa aattcaccct ttattatcaa cagactaaac 96360 ttcctatttt gcgtagatgc tcctgtgcac gcctttattt tacacggaat gtaaaatatt 96420 gtaatgactg attttccttt gagactgtaa tacaggtaat gttcctattc aactttgtaa 96480 accaagacct gacacacagt aggtatttta aaaactgttt tgatgtgacc aaaattatac 96540 agaaaaaaag agaagtacac caggatttta aagtctcttt ttttttttta tttttcacaa 96600 aggatttgct gtaagtcttc aagtcatttt gtccaatcca aaagctgtat ttaagcgtcg 96660 tggatcccag ccagggatgc aagaatctga ctttctcaaa cagataacaa cagtcgaaga 96720 actggaaccg aaagcaaata actgcactaa ggtattcatt acacttgtgc tgcccgacct 96780 cgagtgtcac catgaagagt gcgctaccca agctatttcc ttccccttca ggttctcgtg 96840 tggcacactc ggacagagaa ggttaatcta gccaacgagc caaagtacca cctggacaca 96900 gtgaaaattg aggtataaat tgaagcagca actggtgcag tttgtccagc cagtggatcc 96960 atatggaaga ggatgtttgg agtttaggct acagagcatt caggtattgt ttgttttact 97020 tcagtacagc agcctttctt gtcatctgat ggacatctgt ttaaatggag cttgtcagtt 97080 aacataagct aattggatgg ttggtacaaa atgtatgttt tgtcttcatt tgttctgcat 97140 gttttctcta caacaactaa attggaagat ttttttgtac agtgccgata ctgcaagata 97200 ccactcttga gtatatattt tttctttttc tccaatttgc ccttataatt ggtagacttg 97260 aacaggttgg tagacttgaa caggttttta aaacagacaa gtattttgtc agctaaacgt 97320 tcctgatgat tcctgacttt gcaatactaa gtaatttttg gaaggttagt ggcagtatac 97380 atcataggaa ataaaaaccc acaaatgaaa aggtctatgg agtcatgttt aatgtaggga 97440 aataacattt tgtcaatact aggcaccata aaatgtaaac acaattactg tcataaacct 97500 agatatacct tcaaggattg aagattgaaa gtggctttgt tttagttagt taccctgttt 97560 gcatatagtg cagaaaaagg tcttcatgtt agcactatgt acattaagaa gagatccaaa 97620 ttacaagaga ggcagataaa atttgaattc tttaagcatt cattaaacga agttttggag 97680 taacatccac gtttatcttc ctttcactaa tcacgttccc tgttaagcac atcataacaa 97740 cagcacagtg aagtgaatga tgaaataaga gcattttgat acactagaaa acagtgctca 97800 gtgagacatt tacattctat ttatatgatt aaacatttga tcatacagta ccttcctaca 97860 ggattactgg ctaattttgg ggtggggttt atactattag aggtattact aacatgataa 97920 ctacttccct tatatgcaaa cattagagct ataattttat tgagaggaaa actgattttg 97980 caagttgagc agcttctcaa ataatgcagt acatgaaatc atgggaaata tgagcaaagc 98040 tgcccttgac ataaaatgat ttatcaacct gcttttcacc acatcaaatt gaatcagtac 98100 agaccaacac ggtcaatcag atcattctta atatgaacaa atgggtaaaa agaaaaaaaa 98160 tatgcatatg aataaacagg ggaactagat gcgtttcagc aaggaatgtc aggtggtagt 98220 tctggatgaa acttgtattg cagttttcat ttccacagtt gtgtgctgag agtctgacct 98280 gatgagcttc cagaccatcc tgctgttgtg ctggagggct ggccaaaacc tgcagtaggg 98340 gttgcactac tgatactcat gccagccatc tgctgattca tctgtgaaac atataaaagg 98400 cttagttcaa gaggcttact tcacttttaa ttcttgtttc tttagccaca cagttggtca 98460 ttttttcatt aatgtgacaa ctagtccaag cactggaata aaaacagagt accatacaaa 98520 tatttcttaa agcaaatagc tactttgttc ccttctttat ctactttcta gatacagttt 98580 ccccaaagat taaccacaac ttacttaaaa aaaaatacca aagcaatctt gggattttaa 98640 tgagtccgct actctaacta actttcacct acactaggat attgtgcttt aactactaag 98700 gagtaagaaa attttaggaa gtaaaatagt ctaaaattat cctataaact ttgtatgata 98760 gatattattc tctattaaaa tcttatatac ttcctaaata tttttaaagt ggtcataaag 98820 catttatttc tctcgctgat ctaacaacat aaacatctaa aatttatttt cattgtatgc 98880 aataaagcat aagattacat gtatttttct tcaagactgg agtcaaatat atatatatat 98940 aagcatctta accctgtgat tctcttactt ccaaaattgg tgataagaga aggaaaggca 99000 agatttacca tatagtgagt gggtttaaaa cttacactca gagttagact gtgttcttaa 99060 tttaatacat ttgacttgac ttatttacag tttcaaagac actaacataa actacatcac 99120 taatcaggca taagtgtctg aagaagcaga tcacgtcttc atacctacta aaggacattt 99180 taaccacctt gtcgttggcc agtagattgc actgatggag tgctggagaa cagcatcacc 99240 cttctgcatt atctggaagt aagagccagt attaactcct tcctggttca tctagcacct 99300 taacctgagc tgggtgtgct tcagcatgtt gaccatgtga ctgacactta gcacatacaa 99360 ttttttagat tcccagcggg tagagaccaa tgttttacct atattcttgt aaatggtggt 99420 agcaaaatta actgtgatat atagtgattg tgctaatgtt agaaatcact ctagactatt 99480 ccctgaatgc tctaaaggta aaacaagtga ccaaacagaa accaagattg ccaaaatgct 99540 ggaggaacat caatgggaag tgtaaaagga agaagagtgg gagcatgaac ctctctaaga 99600 gcctttgtct gtgcagctag agaaaagtca gaacacagca cctgaaatag aaatgttcta 99660 tctcagctct aacttaggta gaaataggat tttataatat gaggggatgt ctggttcaca 99720 ccttatggga attgaatctt tttgtactct ttttaaacat aaaagtcatt atagggtatg 99780 taaaaagaaa atacaacttt acaaaggttt ctcaacaaaa agaattttta cagagccatg 99840 gggcagtaat catccgacct gaaaaacagc cttagatccc tcataaaata gtgctttgag 99900 aatatgaggc tagatt 99916 4 324 PRT Rattus norvegicus 4 Met Lys Ser Ala Leu Cys Asn Arg Phe Phe Ile Leu Leu Pro Trp Ile 1 5 10 15 Leu Ile Val Ile Ile Met Leu Asp Val Asp Pro Arg Arg Pro Ala Pro 20 25 30 Gln Leu Thr Ser Arg Pro Tyr Phe Ser Pro His Thr Val Gly Cys Gly 35 40 45 Gly Ser Arg Val Pro Leu Arg Arg Ser Ser Pro Gly Arg Asp Ala Ala 50 55 60 Glu Lys Arg Asn Glu Ser Arg Pro Gln Leu Gln Pro Glu Pro Arg Leu 65 70 75 80 Pro Thr Ile Tyr Ala Ile Thr Pro Thr Tyr Ser Arg Pro Val Gln Lys 85 90 95 Ala Glu Leu Thr Arg Leu Ala Asn Thr Phe Arg Gln Val Ala Gln Leu 100 105 110 His Trp Ile Leu Val Glu Asp Arg Ala Thr Arg Ser Glu Leu Val Ser 115 120 125 Ser Phe Leu Ala Arg Ala Gly Leu Pro Asn Thr His Leu His Val Pro 130 135 140 Thr Pro Arg Arg Tyr Lys Arg Pro Trp Leu Pro Arg Ala Thr Glu Gln 145 150 155 160 Arg Asn Ala Gly Leu Ala Trp Leu Arg Gln Arg His Gln His Gln Ser 165 170 175 Ala Gln Pro Gly Val Leu Phe Phe Ala Asp Asp Asp Asn Thr Tyr Ser 180 185 190 Leu Glu Leu Phe Gln Glu Met Arg Thr Thr Arg Lys Val Ser Val Trp 195 200 205 Pro Val Gly Leu Val Gly Gly Arg Arg Tyr Glu Arg Pro Leu Val Lys 210 215 220 Asn Gly Lys Val Val Gly Trp Tyr Thr Gly Trp Arg Glu Asp Arg Pro 225 230 235 240 Phe Ala Ile Asp Met Ala Gly Phe Ala Val Ser Leu Gln Val Ile Leu 245 250 255 Ser Asn Pro Lys Ala Val Phe Lys Arg Arg Gly Ser Gln Pro Gly Met 260 265 270 Gln Glu Ser Asp Phe Leu Lys Gln Ile Thr Thr Val Asp Glu Leu Glu 275 280 285 Pro Lys Ala Asn Asn Cys Thr Lys Val Leu Val Trp His Thr Arg Thr 290 295 300 Glu Lys Val Asn Leu Ala Asn Glu Pro Lys Tyr His Met Asp Thr Val 305 310 315 320 Asn Ile Glu Val 5 197 PRT Homo sapiens 5 Met Lys Ser Ala Leu Phe Thr Arg Phe Phe Ile Leu Leu Pro Trp Ile 1 5 10 15 Leu Ile Val Ile Ile Met Leu Asp Val Asp Thr Arg Arg Pro Val Pro 20 25 30 Pro Leu Thr Pro Arg Pro Tyr Phe Ser Pro Tyr Ala Val Gly Arg Gly 35 40 45 Gly Ala Arg Leu Pro Leu Arg Arg Gly Gly Pro Ala His Gly Thr Gln 50 55 60 Lys Arg Asn Gln Ser Arg Pro Gln Pro Gln Pro Glu Pro Gln Leu Pro 65 70 75 80 Thr Ile Tyr Ala Ile Thr Pro Thr Tyr Ser Arg Pro Val Gln Lys Ala 85 90 95 Glu Leu Thr Arg Leu Ala Asn Thr Phe Arg Gln Val Ala Gln Leu His 100 105 110 Trp Ile Leu Val Glu Asp Ala Ala Ala Arg Ser Glu Leu Val Ser Arg 115 120 125 Phe Leu Ala Arg Ala Gly Leu Pro Ser Thr His Leu His Val Pro Thr 130 135 140 Pro Arg Arg Tyr Lys Arg Pro Gly Leu Pro Arg Ala Thr Glu Gln Arg 145 150 155 160 Asn Ala Gly Leu Ala Trp Leu Arg Gln Arg His Gln His Gln Arg Ala 165 170 175 Gln Pro Gly Val Leu Phe Phe Ala Asp Asp Asp Asn Thr Tyr Ser Leu 180 185 190 Glu Leu Phe Gln Glu 195

Claims

That which is claimed is:

1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence shown in SEQ ID NO:2;

(b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;

(c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and

(d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids.

2. An isolated peptide comprising an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence shown in SEQ ID NO:2;

3. An isolated antibody that selectively binds to a peptide of claim 2.

4. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.

5. A method for producing any of the peptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.

6. A method for detecting the presence of any of the peptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide.

7. A method for identifying a modulator of a peptide of claim 2, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide.

8. The method of claim 7, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide.

9. A method for identifying an agent that binds to any of the peptides of claim 2, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide.

10. A pharmaceutical composition comprising an agent identified by the method of claim 9 and a pharmaceutically acceptable carrier therefor.

11. A method for treating a disease or condition mediated by a human enzyme protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim 9.

12. A method for identifying a modulator of the expression of a peptide of claim 2, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide.

13. An isolated human enzyme peptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence shown in SEQ ID NO:2.

14. A peptide according to claim 13 that shares at least 90 percent homology with an amino acid sequence shown in SEQ ID NO:2.