Patent Publication Number: US-2003224508-A1

Title: Novel vectors and genes exhibiting increased expression

Description:
RELATED APPLICATIONS  
     [0001] This application is a continuation application of Ser. No. 09/553,368, filed on Apr. 20, 2000, which is a divisional application of Ser. No. 09/205,817, filed on Dec. 4, 1998, which claims priority to provisional application serial No. 60/071,596, filed on Jan. 16, 1998, and also claims priority to provisional application serial No. 60/067,614, filed on Dec. 5, 1997. This application also claims priority to PCT application PCT/US98/25354, filed on Nov. 25, 1998. The contents of all of the aforementioned application(s) are hereby incorporated herein by reference. 
    
    
     
       BACKGROUND OF THE INVENTION  
       [0002] Recombinant DNA technology is currently the most valuable tool known for producing highly pure therapeutic proteins both in vitro and in vivo to treat clinical diseases. Accordingly, a vast number of genes encoding therapeutic proteins have been identified and cloned to date, providing valuable sources of protein. The value of these genes is, however, often limited by low expression levels.  
       [0003] This problem has traditionally been addressed using regulatory elements, such as optimal promoters and enhancers, which increase transcription/expression levels of genes. Additional techniques, particularly those which do not rely on foreign sequences (e.g., viral or other foreign regulatory elements) for increasing transcription efficiency of cloned genes, resulting in higher expression, would be of great value.  
       [0004] Accordingly, the present invention provides novel methods for increasing gene expression, and novel genes which exhibit such increased expression.  
       [0005] Gene expression begins with the process of transcription. Factors present in the cell nucleus bind to and transcribe DNA into RNA. This RNA (known as pre-mRNA) is then processed via splicing to remove non-coding regions, referred to as introns, prior to being exported out of the cell nucleus into the cytoplasm (where they are translated into protein). Thus, once spliced, pre-mRNA becomes mRNA which is free of introns and contains only coding sequences (i.e., exons) within its translated region.  
       [0006] Splicing of vertebrate pre-mRNAs occurs via a two step process involving splice site selection and subsequent excision of introns. Splice site selection is governed by definition of exons (Berget et al. (1995)  J. Biol. Chem.  270(6):2411-2414), and begins with recognition by splicing factors, such as small nuclear ribonucleoproteins (snRNPs), of consensus sequences located at the 3′ end of an intron (Green et al. (1986)  Annu. Rev. Genet.  20:671-708). These sequences include a 3′ splice acceptor site, and associated branch and pyrimidine sequences located closely upstream of 3′ splice acceptor site (Langford et al. (1983)  Cell  33:519-527). Once bound to the 3′ splice acceptor site, splicing factors search downstream through the neighboring exon for a 5′ splice donor site. For internal introns, if a 5′ splice donor site is found within about 50 to 300 nucleotides downstream of the 3′ splice acceptor site, then the 5′ splice donor site will generally be selected to define the exon (Robberson et al. (1990)  Mol. Cell. Biol.  10(1):84-94), beginning the process of spliceosome assembly.  
       [0007] Accordingly, splicing factors which bind to 3′ splice acceptor and 5′ splice donor sites communicate across exons to define these exons as the original units of spliceosome assembly, preceding excision of introns. Typically, stable exon complexes will only form and internal introns thereafter be defined if the exon is flanked by both a 3′ splice acceptor site and 5′ splice donor site, positioned in the correct orientation and within 50 to 300 nucleotides of one another.  
       [0008] It has also been shown that the searching mechanism defining exons is not a strict 5′ to 3′ (i.e., downstream) scan, but instead operates to find the “best fit” to consensus sequence (Robberson et al., supra. at page 92). For example, if a near-consensus 5′ splice donor site is located between about 50 to 300 nucleotides downstream of a 3′ splice acceptor site, it may still be selected to define an exon, even if it is not consensus. This may explain the variety of different splicing patterns (referred to as “alternative splicing”) which is observed for many genes.  
       SUMMARY OF THE INVENTION  
       [0009] The present invention provides novel DNAs which exhibit increased expression of a protein of interest. The novel DNAs also can be characterized by increased levels of cytoplasmic mRNA accumulation following transcription within a cell, and by novel splicing patterns. The present invention also provides expression vectors which provide high tissue-specific expression of DNAs, and compositions for delivering such vectors to cells. The invention further provides methods of increasing gene expression and/or modifying the transcription pattern of a gene. The invention still further provides methods of producing a protein by recombinant expression of a novel DNA of the invention.  
       [0010] In one embodiment, a novel DNA of the invention comprises an isolated DNA (e.g., gene clone or cDNA) containing one or more consensus or near consensus splice sites (3′ splice acceptor or 5′ splice donor) which have been corrected. Such consensus or near consensus splice sites can be corrected by, for example, mutation (e.g., substitution) of at least one consensus nucleotide with a different, preferably non-consensus, nucleotide. These consensus nucleotides can be located within a consensus or near consensus splice site, or within an associated branch sequence (e.g., located upstream of a 3′ splice acceptor site). Preferred consensus nucleotides for correction include invariant (i.e., conserved) nucleotides, including one or both of the invariant bases ( AG ) present in a 3′ splice acceptor site; one or both of the invariant bases ( GT ) present in a 5′ splice donor site; or the invariant  A  present in the branch sequence of a 3′ splice acceptor site.  
       [0011] If the consensus or near consensus splice site is located within the coding region of a gene, then the correction is preferably achieved by conservative mutation. In a particularly preferred embodiment, all possible conservative mutations are made within a given consensus or near consensus splice site, so that the consensus or near consensus splice site is as far from consensus as possible (i.e., has the least homology to consensus as is possible) without changing the coding sequence of the consensus or near consensus splice site.  
       [0012] In another embodiment, a novel DNA of the invention comprises at least one non-naturally occurring intron, either within a coding sequence or within a 5′ and/or 3′ non-coding sequence of the DNA. Novel DNAs comprising one or more non-naturally occurring introns may further comprise one or more consensus or near consensus splice sites which have been corrected as previously summarized.  
       [0013] In a particular embodiment of the invention, the present invention provides a novel gene encoding a human Factor VIII protein. This novel gene comprises one or more non-naturally occurring introns which serve to increase transcription of the gene, or to alter splicing of the gene. The gene may alternatively or additionally comprise one or more consensus splice sites or near consensus splice sites which have been corrected, also to increase transcription of the gene, or to alter splicing of the gene. In one embodiment, the Factor VIII gene comprises the coding region of the full-length human Factor VIII gene, except that the coding region has been modified to contain an intron spanning, overlapping or within-the region of the gene encoding the β-domain. This novel gene is therefore expressed as a β-domain deleted human Factor VIII protein, since all or a portion of the β-domain coding sequence (defined by an intron) is spliced out during transcription.  
       [0014] A particular novel human Factor VIII gene of the invention comprises the nucleotide sequence shown in SEQ ID NO:1. Another particular novel human Factor VIII gene of the invention comprises the coding region of the nucleotide sequence shown in SEQ ID NO:3 (nucleotides 1006-8237). Particular novel expression vectors of the invention comprise the complete nucleotide sequences shown in SEQ ID NOS: 2, 3 and 4. These vectors include novel 5′ untranslated regulatory regions designed to provide high liver-specific expression of human Factor VIII protein.  
       [0015] In still other embodiments, the invention provides a method of increasing expression of a DNA sequence (e.g., a gene, such as a human Factor VIII gene), and a method of increasing the amount of mRNA which accumulates in the cytoplasm following transcription of a DNA sequence. In addition, the invention provides a method of altering the transcription pattern (e.g., splicing) of a DNA sequence. The methods of the present invention each involve correcting one or more consensus or near consensus splice sites within the nucleotide sequence of a DNA, and/or adding one or more non-naturally occurring introns into the nucleotide sequence of a DNA.  
       [0016] In a particular embodiment, the invention provides a method of simultaneously increasing expression of a gene encoding human Factor VIII protein, while also altering the gene&#39;s splicing pattern. The method involves inserting into the coding region of the gene an intron which spans, overlaps or is contained within the portion of the gene encoding the β-domain. The method may additionally or alternatively comprise correcting within either the coding sequence or the 5′ or 3′ untranslated regions of the novel Factor VIII gene, one or more consensus or near consensus splice sites.  
       [0017] In yet another embodiment, the invention provides a method of producing a human Factor VIII protein, such as a β-domain deleted Factor VIII protein, by introducing an expression vector containing a novel human Factor VIII gene of the invention into a host cell capable of expressing the vector, under conditions appropriate for expression, and allowing for expression of the vector to occur. 
     
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
     [0018]FIG. 1 shows the nucleotide sequence of an RNA intron. The  GU  of the 5′ splice donor site, the  AG  of the 3′ splice acceptor site, and the  A  of the Branch are invariant bases (100% conserved and essential for recognition as splice sites). U is T in a DNA intron. The Branch sequence is located upstream from the 3′ splice acceptor site at a distance sufficient to allow for lariat formation during spliceosome assembly (typically within 30-60 nucleotides). N is any nucleotide. Splicing will occur 5′ of the  GT  base pair within the 5′ splice donor site, and 3′ of the  AG  base pair.  
     [0019]FIG. 2 shows the conservative correction of a near consensus 3′ splice acceptor site. The correction is made by silently mutating the A of the invariant (conserved)  AG  base pair to C, G, or T which does not affect the coding sequence of the intron because Ser is encoded by three alternate codons.  
     [0020]FIG. 3 is a map of the coding region of a β-domain deleted human Factor VIII cDNA, showing the positions of the 99 silent point mutations which were made within the coding region (contained in plasmid pDJC) to conservatively correct all near consensus splice sites. Numbering of nucleotides begins with the ATG start coding of the coding sequence. Arrows above the map show positions mutated within near consensus 5′ splice donor sites. Arrows below the map show positions mutated within near consensus 3′ splice acceptor sites. Each “B” shown on the map shows a position mutated within a consensus branch sequence.  
     [0021] FIGS.  4 A- 4 C shows the silent nucleotide substitution made at each of the 99 positions maked by arrows in FIG. 3, as well as the codon containing the substitution and the amino acid encoded.  
     [0022] FIGS.  5 A- 5 O is a comparison of the coding sequence of (a) plasmid pDJC (top) containing the coding region of the human β-domain deleted Factor VIII cDNA modified by making 99 conservative point mutations to correct all near consensus splice sites within the coding region, and (b) plasmid p25D (bottom) containing the same coding sequence prior to making the 99 point mutations. Point mutations (substitions) are indicated by a “v” between the two aligned sequences and correspond to the positions within the pDJC coding sequence shown in FIG. 3. Plasmid p25D contains the same coding region as does plasmid pCY-2 shown in FIG. 7 and referred to throughout the text.  
     [0023]FIG. 6 shows a map of plasmid pDJC including restriction sites used for cloning, regulatory elements within the 5′ untranslated region, and the corrected human γ-domain deleted Factor VIII cDNA coding sequence.  
     [0024]FIG. 7 shows a map of plasmid pCY-2 including restriction sites used for cloning, regulatory elements within the 5′ untranslated region, and the uncorrected (i.e., naturally-occurring) human β-domain deleted Factor VIII cDNA coding sequence. pCY-2 and pDJC are identical except for their coding sequences.  
     [0025]FIG. 8 is a map of the human β-domain deleted Factor VIII cDNA coding region showing the five sections of the cDNA (delineated by restriction sites) which can be synthesized (using overlapping 60-mer oligonucleotides) to contain corrected near consensus splice sites, and then and assembled together to produce a new, corrected coding region.  
     [0026]FIG. 9 is a schematic illustration of the cloning procedure used to insert an engineered intron into the coding region of the human Factor VIII cDNA, spanning a majority of the region of the cDNA encoding the β-domain. PCR fragments were generated containing nucleotide sequences necessary to create consensus 5′ splice donor and 3′ splice acceptor sites when cloned into selected positions flanking the β-domain coding sequence. The fragments were then cloned into plasmid pBluescript and sequenced. Once sequences had been confirmed, the fragments creating the 5′ splice donor (SD) site were cloned into plasmid pCY-601 and pCY-6 (containing the full-length human Factor VIII cDNA coding region) immediatedly upstream of the β-domain coding sequence, and fragments creating the 3′ splice acceptor (SA) site were cloned into pCY-601 and pCY-6 immediately downstream of the β-domain coding sequence. The resulting plasmids are referred to as pLZ-601 and pLZ-6, respectively.  
     [0027]FIG. 10 is a map of the full-length human Factor VIII gene, showing the A1, A2, B, A3, C1 and C2 domains. Following expression of the gene, the β domain is naturally cleaved out of the protein. The map shows the 5′ and 3′ splice sites inserted within the B region of the gene (in plasmid pLZ-6) so that, during pre-mRNA processing of the gene, the majority of the B region will be spliced out. Segments A2 and A3 of the gene will then be juxtaposed, coding for amino acids SFSQNPPV at the juncture.  
     [0028]FIG. 11 shows the nucleotide sequences of the exon/intron boundaries (SEQ ID NO:5) flanking the β-domain coding region in plasmid pLZ-6 (containing the full-length human Factor VIII cDNA). The 5′ splice donor site was added so that splicing would occur 5′ of the “g” shown at position 2290. The 3′ splice acceptor site was added so that splicing would occur 3′ of the “g” shown at position 5147. Following splicing of the intron created by these splice sites, amino acids Gln-744 and Asn-1639 of the full-length human Factor VIII protein are brought together, resulting in a deletion of amino acids 745 to 1638 (numbering is in reference to Ala-1 of the mature human Factor VIII protein following cleavage of the 19 amino acid signal peptide). Capital letters represent nucleotide bases which remain within exons of the mRNA. Small case letters represent nucleotide bases which are spliced out of the mRNA as part of the intron.  
     [0029]FIG. 12 is a map of the coding region of the full-length human Factor VIII gene showing (a) ATG (start) and TGA (stop) codons, (b) restriction sites within the coding region, (c) 5′ splice donor (SD) and 3′ splice acceptor (SA) sites of a rabbit β-globin intron positioned upstream of the coding region within the 5′ untranslated region, (d) 5′ splice donor and 3′-splice acceptor sites added within the coding region defining an internal intron spanning the β-domain.  
     [0030]FIG. 13 is a schematic illustration comparing the process of transcription, expression and post-translational modification for human Factor VIII produced from (a) a full-length human Factor VIII gene, (b) a β-domain deleted human Factor VIII gene, and (c) a full-length human Factor VIII gene containing an intron spanning the β-domain coding region.  
     [0031]FIG. 14 is a graphic comparison of human Factor VIII expression for (a) pCY-6 (containing the coding region of the full-length human Factor VIII cDNA, as well as a 5′ untranslated region derived from the second IVS of rabbit beta globin gene), (b) pCY-601 (containing the coding region of the full-length human Factor VIII cDNA, without the rabbit beta globin IVS), (c) pLZ-6 (containing the coding region of a full-length human Factor VIII cDNA with an intron spanning the β-domain, as well as the rabbit beta globin IVS), and (d) pLZ-601 (containing the coding region of a full-length human Factor VIII cDNA with an intron spanning the majority of the β-domain, without the rabbit beta globin IVS). Expression is given in nanograms. Transfection efficiencies were normalized to expression of human growth hormone (hGH). Each bar represents a summary of four separate transfection experiments.  
     [0032]FIG. 15 shows areas within the human Factor VIII transcription unit for sequence optimization.  
     [0033]FIG. 16 shows the optimized intron-split leader sequence within vectors pCY-2, pCY-6, PLZ-6 and pCY2-SRE5, as well as the secondary structure of the leader sequence (SEQ ID NO:11) predicted by the computer program RNAdraw™.  
     [0034]FIG. 17 is a schematic illustration showing two different RNA export pathways. The majority of mRNA&#39;s in higher eukaryotes contain intronic sequences which are removed within the nucleus (splicing pathway), follwed by export of the mRNA into the cytoplasm. Mammalian intronless genes, hepadnaviruses (e.g., HBV), and many retroviruses access a nonsplicing pathway which is facilitated by cellular RNA export proteins (facilitated pathway).  
     [0035]FIG. 18 is a graph showing the effect of a 5′ intron and 3′ post-transcriptional regulatory element (PRE) on human Factor VIII expression levels in HuH-7 cells. Plasmid pCY-2 contains a 5′ intron but no PRE. Plasmid pCY-201 is identical to pCY-2, except that it lacks the 5′ intron. Plasmid pCY-401 and pCY-402 are identical to pCY-201, except that they contain one and two copies of the PRE, respectively. The levels of secreted active Factor VIII was measured from supernatants collected 48 hours (first bar of each group) or 72 hours (second bar of each group) after transfection by Coatest VIII: c/4 kit from Kabi Inc. The transfection efficiency of each plasmid was normalized by analysis of human growth hormone secreted levels.  
     [0036]FIG. 19 is a graph comparing human Factor VIII expression in vivo in mice for plasmids containing various regulatory elements upstream of either the β-domain deleted or full-length human Factor VIII gene. Plasmid pCY-2 has a 5′ untranslated region containing the liver-specific thyroxin binding globulin (TBG) promoter, two copies of the liver-specific alpha-1 microglobulin/bikunin (ABP) enhancer; and a modified rabbit β-globin IVS, all upstream of the human β-domain deleted Factor VIII gene. Plasmid pCY2-SE5 is identical to pCY-2 except that the TBG promoter was replaced by the endothelium-specific human endothelin-1 (ET-1) gene promoter, and the ABP enhancers (both copies) were replaced by one copy of the human c-fos gene (SRE) enhancer. Plasmid pCY-6 is identical to pCY-2, except that the human β-domain deleted Factor VIII gene was replaced by the full-length human Factor VIII gene. Plasmid pLZ-6 is identical to pCY-6, except that the full-length human Factor VIII gene contained an intron spanning the β-domain. Plasmid pLZ-6A is identical to pLZ-6, except that it contains one corrected near consensus 3′ splice acceptor site (A to C at base 3084 of pCY-6 (SEQ ID NO:3). Each bar represents an average of five mice.  
     [0037]FIG. 20 shows the nucleotide sequence of the human alpha-1 microglobulin/bikunin (ABP) enhancer. Clustered liver-specific elements are underlined and labeled HNF-1, HNF-3 and HNF-4.  
     [0038]FIG. 21 shows the nucleotide sequence of the human thyroxin binding globulin (TBG) promoter, also containing clustered liver-specific enhancer elements.  
     [0039]FIG. 22 shows the nucleotide sequence and secondary structure of an optimized leader sequence.  
     [0040]FIG. 23 is a comparison of the nucleotide sequences of the rabbit β-globin IVS before (top line) and after, (bottom line) optimization to contain consensus 5′ splice donor, 3′ splice acceptor, branch, and translation initiation sites. Five nucleotides were also changed from purines to pyrimidines to optimize the pyrimidine track.  
     [0041]FIG. 24 contains a list of various endothelium-specific promoters and enhancers, and characteristics associated with these promoters and enhancers.  
     [0042]FIG. 25 is a graph comparing expression of plasmid pCY-2 and p25D in vivo in mice. Both plasmids contain the same coding sequence (for human β-domain deleted Factor VIII). Plasmid pCY-2 has an optimized 5′ UTR containing two copies of the ABP enhancer, one copy of the TBG promoter and a leader sequence split by an optimized 5′ rabbit β-globin intron. Plasmid p25D has a 5′ UTR containing one copy of the CMV enhancer, one copy of the CMV promoter, and a leader sequence containing a short (130 bp) chimeric human IgE intron. Each bar represents an average of 5 mice. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0043] Definitions  
     [0044] The present invention is described herein using the following terms which shall be understood to have the following meanings:  
     [0045] An “isolated DNA” means a DNA molecule removed from its natural sequence context (i.e., from its natural genome). The isolated DNA can be any DNA which is capable of being transcribed in a cell, including for example, a cloned gene (genomic or cDNA clone) encoding a protein of interest, operably linked to a promoter. Alternatively, the isolated DNA can encode an antisense RNA.  
     [0046] A “5′ consensus splice site” means a nucleotide sequence comprising the following bases: MAG GT RAGT, wherein M is (C or A), wherein R is (A or G) and wherein  GT  is essential for recognition as a 5′ splice site (hereafter referred to as the “essential  GT  pair” or the “invariant  GT  pair”).  
     [0047] A “3′ consensus splice site” means a nucleotide sequence comprising the following bases (Y&gt;8)NY AG G, wherein Y&gt;8 is a pyrimidine track containing at least eight (most commonly twelve to fifteen or more) tandem pyrimidines (i.e., C or T (U if RNA)), wherein N comprises any nucleotide, wherein Y is a is a pyrimidine, and wherein the  AG  is essential for recognition as a 3′ splice site (hereafter referred to as the “essential  AG  pair” or the “invariant  AG  pair”). A “3′ consensus splice site” is also preceded upstream (at a sufficient distance to allow for lariat formation, typically at least about 40 bases) by a “branch sequence” comprising the following seven nucleotide bases: YNYTR A Y, wherein Y is a pyrimidine (C or T), N is any nucleotide, R is a purine (A or G), and A is essential for recognition as a branch sequence (hereafter referred to as “the essential  A ” or the “invariant  A ”). When all seven branch nucleotides are located consecutively in a row, the branch sequence is a “consensus branch sequence.”A “near consensus splice site” means a nucleotide sequence which:  
     [0048] (a) comprises the essential 3′  AT  pair, and is at least about 50% homologous, more preferably at least about 60-70% homologous, and most preferably greater than 70% homologous to a 3′ consensus splice site, when aligned with the consensus splice site for purposes of comparison; or  
     [0049] (b) comprises the essential 5′  GT  pair, and is at least about 50% homologous, more preferably at least about 60-70% homologous, and most preferably greater than 70% homologous to a 5′ consensus splice site, when aligned with the consensus splice site for purposes of comparison.  
     [0050] Homology refers to sequence similarity between two nucleic acids. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.  
     [0051] As will be described in more detail below, additional criteria for selecting “near consensus splice sites” can be used, adding to the definition provided above. For example, if a near consensus splice site shares homology with a 5′ consensus splice site in only 5 out of 9 bases (i.e., about 55% homology), then these bases can be required to be located consecutively in a row. It can additionally or alternatively be required that a 3′ near consensus splice site be preceded by a consensus branch sequence (i.e., no mismatches allowed), or followed downstream by a consensus or near consensus 5′ splice donor site, to make the selection more stringent.  
     [0052] The term “corrected” as used herein refers to a near consensus splice site mutated by substitution of at least one nucleotide shared with a consensus splice site, hereafter referred to as a “consensus nucleotide”. The consensus nucleotide within the near consensus splice site is substituted with a different, preferably non-consensus nucleotide. This makes the near consensus splice site “farther from consensus.” 
     [0053] If the near consensus splice site is within a coding region of a gene, then the correction is preferably a conservative mutation. A “conservative mutation” means a base mutation which does not affect the amino acid sequence coded for, also known as a “silent mutation.” Accordingly, in a preferred embodiment of the invention, correction of a near consensus splice site located within the coding region of a gene includes making all possible conservative mutations to consensus nucleotides within the site, so that the near consensus splice site is as far from consensus as possible without changing the amino acid sequence it encodes.  
     [0054] A “Factor VIII gene” as used herein means a gene (e.g., a cloned genomic gene or a cDNA) encoding a functional human Factor VIII protein from any species (e.g., human or mouse). A Factor VIII gene which is “full-length” comprises the complete coding sequence of the human Factor VIII gene found in nature, including the region encoding the β-domain. A Factor VIII gene which “encodes a β-domain deleted Factor VIII protein” or “a β-domain deleted Factor VIII gene” lacks all or a portion of the region of the full-length gene encoding the β-domain and, therefore, is transcribed and expressed as a “truncated” or “β-domain deleted” Factor VIII protein. A gene which “is expressed as a β-domain deleted Factor VIII protein” includes not only a gene which encodes a β-domain deleted Factor VIII protein, but also a novel Factor VIII gene provided by the present invention which comprises the coding region of a full-length Factor VIII gene, except that it additionally contains an intron spanning the portion of the gene encoding the β-domain. The term “spans” means that the intron overlaps, encompasses, or is encompassed by the portion of the gene encoding the β domain. The portion of the gene spanned by the intron is then spliced out of the gene during transcription, so that the resulting mRNA is expressed as a truncated or β-domain deleted Factor VIII protein.  
     [0055] A “truncated” or “β-domain deleted” Factor VIII protein includes any active Factor VIII protein (human or otherwise) which contains a deletion of all or a portion of the β-domain.  
     [0056] A “non-naturally occurring intron” means an intron (defined by a 5′ splice donor site and a 3′ splice acceptor site) which has been engineered into a gene, and which is not present in the natural DNA or pre-mRNA nucleotide sequences of the gene.  
     [0057] An “expression vector” means any DNA vector (e.g., a plasmid vector) containing the necessary genetic elements for expression of a novel gene of the present invention. These elements, including a suitable promoter and preferably also a suitable enhancer, are “operably linked” to the gene, meaning that they are located at a position within the vector which enables them to have a functional effect on transcription of the gene.  
     [0058] Identification of Consensus and Near Consensus Splice Sites  
     [0059] A consensus or near consensus splice site can be identified within a DNA, or its corresponding RNA transcript, by evaluating the nucleotide sequence of the DNA for the presence of a sequence which is identical or highly homologous to either a 3′ consensus splice acceptor site or a 5′ consensus splice donor site (FIG. 1). Such consensus and near consensus sites can be located within any portion of a given DNA (e.g., a gene), including the coding region of the DNA and any 3′ and 5′ untranslated regions.  
     [0060] To identify 3′ consensus and near consensus splice acceptor sites, a DNA (or corresponding RNA) sequence is analyzed for the presence of one or more nucleotide sequences which includes an  AG  base pair, and which is either identical to or at least about 50% homologous, more preferably at least about 60-70% sequence homologous, to the sequence: (T/C)≧8 N(C/T) AG G. In a preferred embodiment, the nucleotide sequence is also followed upstream, typically by about 40 bases, by a nucleotide sequence which is identical to or highly homologous (e.g., at least about 50%-95% homologous) to a branch consensus sequence comprising the following bases: (C/T)N(C/T)T(A/G) A (C/T), wherein N is any nucleotide, and  A  is invariant (i.e., essential). By way of example, in studies described herein, consensus and near consensus 3′ splice sites were selected for correction within a gene encoding Factor VIII using the following criteria: the consensus or near consensus site (a) contained an  AG  pair, and (b) contained no more than three mismatches to a 3′ consensus site.  
     [0061] To identify 5′ consensus and near consensus splice donor sites, a DNA (or corresponding RNA) sequence can be analyzed for the presence of one or more nucleotide sequences which contains a  GT  base pair, and which is either identical to or at least about 50% homologous, more preferably at least about 60-70% homologous, to the sequence: (A/C)AG GT (A/G)AGT. By way of example, in studies described herein, consensus and near consensus 5′ splice sites were selected for correction within a gene encoding Factor VIII using the following criteria: the consensus or near consensus site (a) contained a  GT  pair, and (b) contained no more than four mismatches to a 5′ consensus site, provided that if it contained four mismatches, they were located consecutively in a row.  
     [0062] Evaluation of DNA or RNA sequences for the presence of one or more consensus or near consensus splice sites can be performed in any suitable manner. For example, nucleotide sequences can be manually analyzed. Alternatively, a computer algorithm can be employed to search nucleotide sequences for specified base patterns (e.g., the MacVector™ program). The latter approach is preferred for large DNAs or RNAs, particularly because it allows for easy implementation of multiple search parameters.  
     [0063] Correction of Consensus and Near Consensus Splice Sites  
     [0064] In one embodiment of the invention, splice and branch sequences which are consensus, or near consensus, are corrected by substitution of one or more consensus nucleotides within the site. The consensus nucleotide within the site is preferably substituted with a non-consensus nucleotide. For example, if the nucleotide being substituted is a C (i.e., a pyrimidine) and the consensus sequence contains either C or T, then the nucleotide is preferably substituted by an A or G (i.e., a purine), thereby making the consensus or near consensus splice site “farther from consensus.” 
     [0065] In a preferred embodiment of the invention, consensus and near consensus sites which are located within a coding region of a gene are corrected by conservative substitution of one or more nucleotides so that the correction does not affect the amino acid sequence coded for. Such conservative or “silent” mutation of codons to preserve coding sequences is well known in the art. Accordingly, the skilled artisan will be able to select appropriate base substitutions to retain the coding sequence of any codon which forms all or part of a consensus or near consensus splice site. For example, as shown in FIG. 2, if a 3′ near consensus splice site contains a  TCA  codon encoding serine, and the  A  is a consensus nucleotide (e.g., part of the essential  AG  pair, then this nucleotide can be substituted with a C, G, or a T to correct the 3′ near consensus splice site (e.g., making it no longer near consensus because it does not contain the essential  AG  pair required for a 3′ near consensus splice site), without affecting the coding sequence of the codon.  
     [0066] Accordingly, in a preferred embodiment of the invention, correction of consensus or near consensus splice sites which are specifically located within the coding region of a gene is achieved by substitution of one or both bases of an essential  AG  or  GT  pair within the consensus or near consensus splice site, with a base which does not alter the coding sequence of the site. Correction of consensus or near consensus branch sequences is similarly achieved by substitution of the essential  A  within the consensus or near consensus branch site, with a base which does not alter the coding sequence of the site. By correcting any of these essential bases, the splice or branch site will no longer be consensus or near consensus.  
     [0067] In another preferred embodiment, correction of consensus or near consensus splice sites which are specifically located within the coding region of a gene is achieved by making all possible conservative mutations to consensus nucleotides within the site, so that the consensus or near consensus splice site is as far from consensus as possible but encodes the same amino acid sequence.  
     [0068] Other preferred corrections of the invention include corrections of 3′ consensus and near consensus splice sites which are followed downstream (e.g., by approximately 50-350 nucleotides) by a consensus or near consensus 5′ splice donor site. Other preferred corrections of the invention include corrections of 5′ consensus and near consensus splice sites which are preceded upstream (e.g., by about 50-350 nucleotides) by a consensus or near consensus 3′ splice acceptor site.  
     [0069] For consensus or near consensus splice sites which are located outside the coding region of a gene, for example, in a 3′ or 5′ untranslated region (UTR), alternative approaches to correction can also be employed. For instance, because preservation of the coding sequence is not a consideration, the near consensus splice site can be corrected not only by any base substitution, but also by addition or deletion of one or more bases within the consensus or near consensus splice site, making the site farther from consensus.  
     [0070] Techniques for making nucleotide base substitutions, additions and deletions as described above are well known in the art. For example, standard point mutation may be employed to substitute one or more bases within a near consensus splice site with a different (e.g., non-consensus) base. Alternatively, as described in detail in the examples below, entire genes or portions thereof can be reconstructed (e.g., resynthesized using PCR), to correct multiple consensus and near consensus splice sites within a particular region of a gene. This approach is particularly advantageous if a gene contains a high concentration of consensus and/or near consensus splice sites within a given region.  
     [0071] In a specific embodiment, the invention features a novel Factor VIII gene containing one or more consensus or near consensus splice sites which have been corrected by substitution of one or more consensus nucleotides within the site. As part of the present invention, the coding region of a gene (cDNA) encoding human β-domain deleted Factor VIII protein (nucleotides 1006-5379 of SEQ ID NO:2) was evaluated as described herein and found to contain 23 near consensus 5′ splice (donor) sequences, 22 near consensus 3′ splice (acceptor) sequences, and 18 consensus branch sequences (shown in FIG. 3). A new coding sequence (SEQ ID NO:1) was then developed for this gene to correct all 3′ and 5′ near consensus splice sites by conservative mutation. In total, 99 point mutations were made to the coding region. The location of each of these point mutations is shown in FIG. 3. The specific base substitution made in each of these point mutations is shown in FIG. 4(A-C).  
     [0072] A comparison of this new coding sequence (SEQ ID NO:1) and the original uncorrected sequence (nucleotides 1006-5379 of SEQ ID NO:2), also showing the positions and specific substitutions made in each of the ninety-nine point mutations, is shown in FIG. 5(A-O). A plasmid vector, referred to as pDJC, containing the new (i.e., corrected) Factor VIII gene coding sequence, including restriction sites used to synthesize the gene and regulatory elements used to express the gene, is shown in FIG. 6. A plasmid vector, referred to as pCY2, containing the original, uncorrected Factor VIII gene, including restriction sites and regulatory elements used to express the gene, is shown in FIG. 7.  
     [0073] As described in further detail in the examples below, all 99 consensus base corrections within the coding region of pDJC can be made by synthesizing overlapping oligonucleotides (based on the sequence of pCY2 shown in SEQ ID NO:2) which contain the desired corrections. A schematic illustration of this process is shown in FIG. 8. In total, 185 overlapping 60-mer oligonucleotides can be synthesized, and assembled in five segments using the method of Stemmer et al. (1995)  Gene  164: 49-53. Prior to assembly, each segment can be sequenced and tested in in vitro transfection assays (e.g., nuclear and cytoplasmic RNA analysis) in pCY2.  
     [0074] As an alternative to the “correct all” approach described above, selective correction of consensus and near consensus splice sites can also be employed. This involves selecting only (a) consensus sites, and near consensus splice sites which are close to consensus, and/or (b) consensus sites and near consensus sites which are located at positions which render these sites more likely to function as a splice donor or acceptor site. To select only nucleotide sequences which are complete consensus or which are close to consensus, evaluation of a given nucleotide sequence is limited to analyzing the nucleotide sequence for sequences which are identical to or are highly homologous (e.g., greater than 70-80% homologous) to a 3′ or 5′ consensus splice site. To select only nucleotide sequences which are located at positions which render these sites more likely to function as a splice donor or acceptor site, the location of each 3′ consensus or near consensus splice site must be evaluated with respect to the position of any neighboring 5′ consensus or near consensus splice sites. If a 3′ consensus or near consensus splice site is located approximately 50-350 bases upstream from a 5′ consensus or near consensus splice site, then these 3′ and 5′ splice sites are likely to function as a splice acceptor and donor sites. Therefore, these sites are preferably, and selectively, removed.  
     [0075] By way of example, particular consensus and/or near consensus 5′ splice donor and 3′ splice acceptor sites, as shown in FIG. 3, can be selected within the coding region of the cDNA encoding human β-domain deleted Factor VIII (nucleotides 1006-5379 of SEQ ID NO:2) for preferred correction, based on their relative locations (i.e., 3′ splice acceptor site located approximately 50-350 bases upstream from 5′ near consensus splice site). Such preferred selective corrections can include, for instance, the near consensus 3′ splice acceptor site spanning nucleotide base 1851 of the coding region (see FIG. 3) and any of the near consensus 5′ splice donor sites located within 50-350 bases downstream of this near consensus 3′ splice acceptor site, such as those spanning positions 1956, 1959, 2115, 2178 and 2184.  
     [0076] Splice site correction as provided herein can be applied to any gene known in the art. For example, the complete nucleotide sequence of other (e.g., full-length and β-domain deleted) Factor VIII genes (both genomic clones and cDNAs) are described in U.S. Pat. No. 4,757,006, U.S. Pat. No. 5,618,789, U.S. Pat. No. 5,683,905, and U.S. Pat. No. 4,868,112, the disclosures of which are incorporated by reference herein. The nucleotide sequences of these genes can be analyzed for consensus and near consensus splice sites, and thereafter corrected, using the guidelines and procedures provided herein.  
     [0077] In addition, other genes, particularly large genes containing several introns and exons, are also suitable candidates for splice site correction. Such genes, include, for example, the gene encoding Factor IX, or the cystic fibrosis transmembrane regulator (CFTR) gene described in U.S. Pat. No. 5,240,846, or nucleic acids encoding CFTR monomers, as described in U.S. Pat. No. 5,639,661. The disclosures of both of these patents are accordingly incorporated by reference herein.  
     [0078] Addition of Introns  
     [0079] In another embodiment, a novel gene of the invention includes one or more non-naturally occurring introns which have been added to the gene to increase expression of the gene, or to alter the splicing pattern of the gene. The present invention provides the first known instance of gene engineering which involved adding a non-naturally-occurring intron within the coding sequence of a gene, particularly without affecting the activity of the protein encoded by the gene. The benefit of intron addition in this context is at least two-fold. First, as shown in FIG. 14 in the context of the human Factor VIII gene, addition of one or more introns into a gene increases the expression of the gene compared to the same gene without the intron. Second, the intron, when placed within the coding sequence of the gene, can be used to beneficially alter the splicing pattern of the gene (e.g., so that a particular protein of interest is expressed), and/or to increase cytoplasmic accumulation of mRNA transcribed from the gene.  
     [0080] Novel genes of the present invention may also contain introns outside of the coding region of the gene. For example, introns may be added to the 3′ or 5′ non-coding regions of the gene (utranslated regions (UTRs)). In a preferred embodiment of the invention, an intron is added upstream of the gene in the 5′ UTR, as shown in pDJC (FIG. 6) and pCY2 (FIG. 7). Such introns may include newly engineered introns or pre-existing introns. In a preferred embodiment of the invention, the intron is derived from the rabbit β-globin intron (IVS).  
     [0081] In a particular embodiment, the invention provides a novel human Factor VIII gene which includes within its coding region one or more introns. If the gene comprises the coding region of a full-length human Factor VIII gene, then at least one of these introns preferably spans (i.e., overlaps, encompasses or is encompassed by) the portion of the gene encoding the β-domain. This portion of the gene is then spliced out during transcription of the gene, so that the gene is expressed as a β-domain deleted protein (i.e., a Factor VIII protein lacking all or a portion of the β-domain).  
     [0082] A β-domain deleted human Factor VIII protein possesses known advantages over a full-length human Factor VIII protein (also known as human Factor VIII:C), including reduced immunogenicity (Toole et al. (1986)  PNAS  83:5939-5942). Moreover, it is well known that the β-domain is not needed for activity of the Factor VIII protein. Thus, a novel Factor VIII gene of the invention provides the dual benefit of (1) increased and (2) preferred protein expression.  
     [0083] Addition of one or more introns into a gene can be achieved by adding a 5′ splice donor site and a 3′ splice acceptor site (FIG. 1) into the nucleotide sequence of the gene at a desired location. If the intron is being added to remove a portion of the coding sequence from the gene, then a 5′ splice donor site is placed at the 5′ end of the portion being removed (i.e., defined by the intron) and a 3′ splice acceptor site is placed at the 3′ end of the portion to be removed. Preferably, the 5′ splice donor and 3′ splice acceptor sequences are consensus, including the branch sequence located upstream of the 3′ splice site, so that they will be favored (and more likely bound) by cellular splicing machinery over any surrounding near consensus splice sites.  
     [0084] As shown in FIG. 1, splicing will occur 5′ of the essential  GT  base pair within the 5′ splice donor site, and 3′ of the essential  AG  base pair within the 3′ splice acceptor site. Thus, for introns added to coding sequences of genes, the intron is preferably designed to that, upon splicing, the coding sequence is unaffected. This can be done by designing and adding 5′ splice donor and 3′ splice acceptor sites which include only conservative (i.e., silent) changes to the nucleotide sequence of the gene, so that addition of these splice sites does not alter the coding sequence.  
     [0085] For example, as part of the present invention, an intron was engineered into the coding sequence of a full-length cDNA encoding human Factor VIII (1006-8061 of SEQ ID NO:4). The intron spanned the portion of the gene encoding the β-domain (nucleotides 2290-5147 of SEQ ID NO:4, encoding amino acid residues 745-1638). As described in the examples below, this intron was created by adding a 5′ splice donor site (100% consensus) so that splicing would occur immediately 5′ of the coding sequence of the β-domain. A 3′ splice acceptor site was also added so that splicing would occur immediately 3′ of the coding sequence of the β-domain. FIG. 11 shows the nucleotide sequences (SEQ ID NO:5) of the precise boundaries of the resulting intron that was added.  
     [0086] The nucleotide sequence for the 5′ splice donor site of the added intron was derived from the pre-existing splice donor sequence found at the 5′ end of IVS (Intron) 13 of genomic Factor VIII. This intron precedes exon 14, the exon which contains the sequence coding for the β-domain. The inserted sequence also contained the first nine bases of IVS 13 following the splice donor sequence.  
     [0087] The sequence for the 3′ splice acceptor site was derived from the pre-existing splice acceptor sequence found at the 3′ end of IVS 14 of genomic Factor VIII. This intron follows exon 14, the β-domain-containing exon. The inserted 3′ splice acceptor site also contained 130 bases upstream of the splice acceptor in IVS 14. This upstream region contains at least two near-consensus branch sequences.  
     [0088] Thus, both the 3′ and 5′ engineered splice sites were designed to take advantage of pre-existing nucleotide sequences within the β-domain region of the human Factor VIII gene.  
     [0089] The 5′ splice donor, 3′ splice acceptor, and branch sequences of the added intron were further modified so that they were 100% consensus (i.e., congruent to their respective consensus splicing sequences). Modifications (e.g., base substitutions) were chosen so as to not alter the coding sequence of bases located upstream of the 5′ splice site and downstream of the 3′ splice site (i.e., flanking the boundaries of the intron). A map showing the various domains of the full-length Factor VIII gene, along with the 5′ splice donor and 3′ splice acceptor sites inserted into the gene, is shown in FIG. 10. The complete nucleotide sequences of the intron boundaries (i.e., 5′ splice donor and 3′ splice acceptor) are shown in FIG. 11 (SEQ ID NO:5). A map showing the location of the location of the 5′ splice donor and 3′ splice acceptor sites with respect to various restriction sites (used to clone in the sites) is shown in FIG. 12. As shown schematically in FIG. 13, the resulting novel Factor VIII gene, in contrast to a full-length Factor VIII gene or a gene encoding β-domain deleted Factor VIII, is transcribed as a pre-mRNA which contains the region encoding the β-domain, but is then spliced to remove the majority of this region, so that the resulting mRNA is expressed as a β-domain deleted protein. A complete expression plasmid (pLZ-6) containing the coding sequence of this novel Factor VIII gene, as well as an engineered 5′ untranslated region containing regulatory elements designed to provide high, liver-specific expression, comprises the nucleotide sequence shown in SEQ ID NO:3. Bases 1006-8237 of pLZ-6 (SEQ ID NO:3) correspond to the coding region of the novel Factor VIII gene.  
     [0090] Accordingly, in a preferred embodiment, the invention provides a novel Factor VIII gene comprising a non-naturally occurring intron spanning all or a portion of the β-domain region of the gene. In one embodiment, the gene comprises the coding region of the nucleotide sequence shown in SEQ ID NO:3. The gene may also contain further modifications, such as additional introns, or one or more corrected consensus or near consensus splice sites as described herein. In particular, the gene may further comprise one or more introns upstream of the coding sequence of the gene, within the 5′ UTR. As shown in FIGS. 6 and 7, a preferred intron for insertion within this region is the rabbit β-globin intron (IVS). In addition, consensus and near consensus splice site corrections can be made to the gene, such as those shown in FIGS. 3 and 4(A-C).  
     [0091] Optimization of 5′ and 3′ Untranslated Regions for High Tissue-Specific Gene Expression  
     [0092] Novel DNAs of the invention are preferably in a form suitable for transcription and/or expression by a cell. Generally, the DNA is contained in an appropriate vector (e.g., an expression vector), such as a plasmid, and is operably linked to appropriate genetic regulatory elements which are functional in the cell. Such regulatory sequences include, for example, enhancer and promoter sequences which drive transcription of the, gene. The gene may also include appropriate signal and polyadenylation sequences which provide for trafficking of the encoded protein to intracellular destinations or export of the mRNA. The signal sequence may be a natural sequence of the protein or an exogenous sequence.  
     [0093] Suitable DNA vectors are known in the art and include, for example, DNA plasmids and transposable genetic elements containing the aforementioned genetic regulatory and processing sequences. Particular expression vectors which can be used in the invention include, but are not limited to, pUC vectors (e.g., pUC19) (University of California, San Francisco) pBR322, and pcDNA1 (InVitrogen, Inc.). An expression plasmid, pMT2LA8, encoding a β-domain deleted Factor VIII protein is described, for example, by Pitman et al. (1993)  Blood  81(11):2925-2935). Entire coding sequences for these plasmid vectors are also provided herein (SEQ ID NOS: 4 and 2, respectively).  
     [0094] Suitable regulatory sequences required for gene transcription, translation, processing and secretion are art-recognized, and are selected to direct expression of the desired protein in an appropriate cell. Accordingly, the term “regulatory sequence”, as used herein, includes any genetic element present 5′ (upstream) or 3′ (downstream) of the translated region of a gene and which control or affect expression of the gene, such as enhancer and promoter sequences (e.g., viral promoters, such as SV40 and CMV promoters). Such regulatory sequences are discussed, for example, in Goeddel,  Gene expression Technology: Methods in Enzymology , page 185, Academic Press, San Diego, Calif. (1990), and can be selected by those of ordinary skill in the art for use in the present invention.  
     [0095] In a preferred embodiment of the invention, the 5′ and/or 3′ untranslated regions (UTRs) of a gene construct (e.g., a novel DNA of the invention) are optimized to provide high, tissue-specific expression. Such optimization can include, for example, selection of optimal tissue-specific promoters and enhancers, multerimization of genetic elements, insertion of one or more introns within or outside of the coding sequence, correction of near-consensus 5′ splice donor and 3′ splice acceptor sites within or outside of the coding sequence, optimization of transcription initiation and termination sites, insertion of RNA export elements, and addition of polyadenylation trimer cassettes to insulate transription. In preferred embodiments of the invention, a combination of the aforementioned elements and sequence modifications are selected and engineered into the gene construct to provide optimized expression.  
     [0096] For many applications of human gene therapy, it is desirable to express proteins in the liver, which has the highest rate of protein synthesis per gram of tissue. For example, effective gene therapy for human Factor VIII requires sufficient levels and duration of protein expression in hepatocytes where Factor VIII is naturally produced, and/or in endothelial cells (ECs) where von Willebrand factor is produced, a protein which stabilizes the secretion of Factor VIII. Thus, in one embodiment, the invention provides a gene construct (e.g., expression vector) optimized to produce high levels and duration of liver-specific protein expression. In a particular embodiment, the invention provides a human Factor VIII gene construct, optimized to produce high levels and duration of liver-specific or endothelium-specific protein expression. This is achieved, for example, by selecting optimal liver-specific and endothelium-specific promoters and enhancers, and by combining these tissue-specific elements with other genetic elements and modifications to increase gene transcription.  
     [0097] Accordingly, for high levels and duration of gene expression in the liver, suitable promoters include, for example, promoters known to contain liver-specific elements. In one embodiment, the invention employs the thyroid binding globulin (TBG) promoter described by Hayashi et al. (1993)  Molec. Endocrinol.  7:1049-1060. As shown in FIG. 21, the TBG promoter contains hepatic nuclear factor (HNF) enhancer elements and provides the additional advantage of having a precisely mapped transcriptional start site. This allows insertion of a leader sequence, preferably optimized as described herein, between the promoter and the transcriptional start site. FIG. 21 also shows the complete nucleotide sequence of the TBG promoter (SEQ ID NO:10).  
     [0098] For high levels and duration of gene expression in endothelium, suitable endothelium-specific promoters include, for example, the human endothelin-1 (ET-1) gene promoter described by Lee et al. (1990)  J. Biol. Chem.  265(18), the fms-like tyrosine kinase promoter (Flt-1) described by Morishita et al. (1995)  J. Biol. Chem.  270(46), the Tie-2 promoter described by Korhonen et al. (1995)  Blood  86(5):1828-1835, and the nitric oxide synthase promoter described by Zhang et al. (1995)  J. Biol. Chem.  270(25)) (see FIG. 24).  
     [0099] Promoters selected for use in the invention are preferably paired with a suitable ubiquitous or tissue-specific enhancer designed to augment transcription levels. For example, in one embodiment, a liver-specific promoter, such as the TBG promoter, is used in conjunction with a liver-specific enhancer. In a preferred embodiment, the invention employs one or more copies of the liver-specific alpha-1 microglobulin/bikunin (ABP) enhancer described by Rouet et al. (1992)  J. Biol. Chem.  267:20765-20773, in combination with the TBG promoter. As shown in FIG. 20, the ABP enhancer contains a cluster of HNF enhancer elements common to many liver-specific genes within a short nucleotide sequence, making it suitable to multerimize. When multerimized, the ABP enhancer generally exhibits increased activity and functions in either orientation within a gene construct.  
     [0100] Thus, in one embodiment, the invention provides an expression vector or DNA construct comprising one or more copies of a liver-specific or endothelium-specific promoter and a liver-specific or endothelium-specific enhancer, the promoter and enhancer being derived from different genes, such as thyroid binding globulin gene and the alpha-1 microglobulin/bikunin gene.  
     [0101] Alternatively, strong ubiquitous (i.e., non-tissue specific) enhancers can be used in conjunction with tissue-specific promoters, such as the TBG promoter or the ET-1 promoter, to achieve high levels and duration of tissue-specific expression. Such ubiquitous enhancers include, for example, the human c-fos (SRE) gene enhancer described by Treisman et al. (1986)  Cell  46 which, when used in combination with liver-specific promoters (e.g., TBG) or endothelium-specific promoters (e.g., ET-1), provide high levels of tissue-specific expression, as demonstrated in studies described herein.  
     [0102] Accordingly, in a particular embodiment, the invention provides a gene construct which is optimized for specific expression in liver cells by inserting within its 5′ untranslated region one or more copies of the ABP enhancer (preferably two copies) coupled upstream with the TBG promoter, as shown in FIG. 15. Specific gene constructs, such as pCY2 and pDJC, containing these elements inserted upstream of the coding region for human Factor VIII (β-domain deleted and full-length with intron spanning the β-domain), are shown in FIGS. 6 and 7, respectively. In another particular embodiment, the gene construct is optimized for specific expression in endothelial cells by inserting within its 5′ region one or more copies of the c-fos SRE enhancer, or an endothelial-specific enhancer (e.g., the human tissue factor (hTF/m) enhancer described by Parry et al. (1995)  Arterioscler. Thromb. Vasc. Biol.  15:612-621) coupled upstream with the ET-1 promoter.  
     [0103] In addition to selecting optimal promoters and enhancers, optimization of a gene construct can include the use of other genetic elements within the transcriptional unit of the gene to increase and/or prolong expression. In one embodiment, one or more introns (e.g., non-naturally occurring introns) are inserted into the 5′ or 3′ untranslated region (UTR) of the gene. Introns from a broad variety of known genes (e.g., mammalian genes) can be used for this purpose. In one embodiment, the invention employs the first intron (IVS) from the rabbit β-globin gene comprising the nucleotide sequence shown in FIG. 23 (SEQ ID NO:6).  
     [0104] In cases where the intron does not contain consensus 5′ splice donor and 3′ splice acceptor sites, or a consensus branch and pyrimidine track sequence, the intron is preferably optimized (modified) to render these sites completely consensus. This can be achieved, for example, by substituting one or more nucleotides within the 5′ or 3′ splice site, as previously described herein to render the site consensus. For example, when using the rabbit β-globin intron, the nucleotide sequence can be modified as shown in FIG. 16 to render the 5′ splice donor and 3′ splice acceptor sites, and the pyrimidine track, entirely consensus. This can facilitate efficient transcription and export of the gene message out of the cell nucleus, thereby increasing expression. Exemplary nucleotide substitutions within the rabbit β-globin IVS which can be made to achieve this result are shown in FIG. 23 which shows a comparison of the sequence for the unmodified (wild-type) rabbit β-globin intron (SEQ ID NO:6) and the same sequence modified to render the 5′ splice donor and 3′ splice acceptor sites, and the pyrimidine track, entirely consensus (SEQ ID NO:7).  
     [0105] When engineering one or more introns into the 5′ UTR of a gene construct, the intron can be inserted into the leader sequence of the gene, as shown in FIGS. 15, 16 and  22 . Accordingly, the intron can be inserted within the leader sequence, downstream from the promoter and enhancer elements. This can be done in conjunction with one or more additional modifications to the leader sequence, all of which serve to increase transcription, stability and export of mRNAs. Such additional modifications include, for example, optimizing the translation initiation site (Kozak et al. (1986)  Cell  44:283) and/or the secondary structure of the leader sequence (Kozak et al. (1994)  Molec. Biol.  235:95).  
     [0106] Accordingly, in a preferred embodiment, the invention provides a gene construct which contains within its transcriptional unit, one or a combination of the foregoing genetic elements and sequence modifications designed to provide high levels and duration of gene expression, optionally in a tissue-specific manner. In a particular embodiment, the construct contains a gene encoding human Factor VIII (e.g., β-domain deleted or full-length), having a 5′ untranslated region which is optimized to provide significant levels and duration of liver-specific or endothelium-specific expression.  
     [0107] Particularly preferred gene constructs of the invention include, for example, those comprising the nucleotide sequences shown in SEQ ID NO:2 and SEQ ID NO:4, referred to herein respectively as pCY-2 and pLZ-6. These constructs contain the coding sequences for human β-domain deleted Factor VIII (pCY-2) and full-length human Factor VIII (containing an intron spanning the β-domain) (pLZ-6) downstream from an optimized 5′ UTR designed to provide high levels and duration of human Factor VIII expression in liver cells. Other preferred gene constructs comprise the identical 5′ UTR of pCY-2 and pLZ-6, in conjunction with coding sequences for other proteins desired to be expressed in the liver (e.g., other blood coagulation factors, such as human Factor IX).  
     [0108] As shown in FIGS. 7, 15 and  16 , plasmids pCY-2 and pLZ-6 contain 5′ UTRs comprising a novel combination of regulatory elements and sequence modifications shown herein to provide high levels and duration of human Factor VIII expression, both in vitro and in vivo, in liver cells. Specifically, each construct comprises within its 5′ UTR sequentially from 5′ to 3′ (a) two copies of the ABP enhancer (SEQ ID NO:9), (b) one copy of the TBG promoter (SEQ ID NO:10), and (c) an optimized 71 nucleotide leader sequence (SEQ ID NO:11) split by intron 1 of the rabbit β-globin gene. The intron is optimized to contain consensus splice acceptor, donor and pyrimidine track sites.  
     [0109] The leader sequence within the 5′ UTR of pCY-2 and pLZ-6 also contains an optimized translation initiation site (SEQ ID NO: 8). Specifically, the human Factor VIII gene contains a cytosine at the +4 position, following the AUG start codon. This base was changed to a guanine, resulting in an amino acid change within the signal sequence of the protein from a glutamine to a glutamic acid. The leader sequence was further designed to have no RNA secondary structure, as predetermined by an RNA-folding algorithm (FIG. 16) (Kozak et al. (1994) J. Mol. Biol.  235:95).  
     [0110] In addition to optimization of the 5′ UTR of a gene construct, the 3′ UTR can also be engineered to include one or more genetic elements or sequence modifications which increase and/or prolong expression of the gene. For example, the 3′ UTR can be modified to provide optimal RNA processing, export and mRNA stability. In one embodiment of the invention, this is done by increasing translational termination efficiency. In mammalian RNA&#39;s, translational termination is generally optimal if the base following the stop codon is a purine (McCaughan et al. (1995)  PNAS  92:5431). In the case of the human Factor VIII gene, the UGA stop codon is followed by a guanine and is thus already optimal. However, in other gene constructs of the invention which do not naturally contain an optimized translational termination sequence, the termination sequence can be optimized using, for example, site directed mutagenesis, to substitute the base following the stop codon for a purine.  
     [0111] In particular gene constructs of the invention which contain the human Factor VIII gene, the 3′ UTR can further be modified to remove one or more of the three pentamer sequences AUUUA present in the 3′ UTR of the gene. This can increase the stability of the message. Alternatively, the 3′ UTR of the human Factor VIII gene, or any gene having a short-lived messenger RNA, can be switched with the 3′ UTR of a gene associated with a message having a longer lifespan.  
     [0112] Additional modifications for optimizing gene constructs of the invention include insertion of one or more poly A trimer cassettes for optimal polyadenylation and 3′ end formation. These can be inserted within the 5′ UTR or the 3′ UTR of the gene. In a preferred embodiment, the gene construct is flanked on either side by a poly A trimer cassette, as shown in FIG. 15. These cassettes can inhibit transcription originating outside of the desired promoter in the transcriptional unit, ensuring that transcription of the gene occurs only in the tissue where the promoter is active (Maxwell et al. (1989)  Biotechniques  1989 3:276). Additionally, because the poly A trimer cassette functions in both orientations, i.e., on each DNA strand, it can be utilized at the 3′ end of the gene for transcriptional termination and polyadenylation, as well as to inhibit bottom strand transcription and production of antisense RNA.  
     [0113] In further embodiments of the invention, gene optimization includes the addition of viral elements for accessing non-splicing RNA export pathways. The majority of mRNAs in higher eukaryotes contain intronic sequences which are removed within the nucleus, followed by export of the mRNA into the cytoplasm. This is referred to as the splicing pathway. However, as shown in FIG. 17, mammalian intronless genes, hepadnaviruses (e.g., HBV), and many retroviruses access a nonsplicing pathway which is facilitated by cellular RNA export proteins and/or specific sequences within. This is referred to as the facilitated pathway.  
     [0114] In a particular embodiment, the gene construct is modified to include one or more copies of the post-transcriptional regulatory element (PRE) from hepatitis B virus. This 587 base pair element and its function to facilitate export of mRNAs from the nucleus, is described in U.S. Pat. No. 5,744,326. Generally, the PRE element is placed within the 3′ UTR of the gene, and can be inserted as two or more copies to further increase expression, as shown in FIG. 18 (plasmid pCY-401 verses plasmid pCY-402).  
     [0115] Gene constructs (e.g., expression vectors) of the invention can still further include sequence elements which impart both an autonomous replication activity (i.e., so that when the cell replicates, the plasmid replicates as well) and nuclear retention as an episome. Generally, these sequence elements are included outside of the transcriptional unit of the gene construct. Suitable sequences include those functional in mammalian cells, such as the oriP sequence and EBNA-1 gene from the Epstein-Barr virus (Yates et al. (1985)  Nature  313:812). Other suitable sequences include the  E. coli  origen of replication, as shown in FIGS. 6 and 7.  
     [0116] Gene constructs of the invention, such as pDJC, pCY-2, pCY-6, pLZ-6 and pCY2-SE5, have been described above, but are not intended to be limiting. Other novel constructs can be made in accordance with the guidelines provided herein, and are intended to be included within the scope of the present invention.  
     [0117] Increased Cytoplasmic RNA Accumulation and Expression  
     [0118] Novel DNAs (e.g., genes) of the present invention are modified to increase expression, for example, by facilitate cytoplasmic accumulation of mRNA transcribed from the DNA and by optimizing the 5′ and 3′ untranslated regions of the DNA. Accordingly, cytoplasmic mRNA accumulation and/or expression of the DNA is increased relative to the same DNA in unmodified form.  
     [0119] To evaluate (e.g., quantify) levels of nuclear or cytoplasmic mRNA accumulation obtained following transcription of novel DNAs and vectors of the invention, a variety of art recognized techniques can be employed, such as those described in Sambrook et al. “Molecular Cloning,” 2d ed., and in the examples below. Such techniques include, for instance, Northern blot analysis, using total nuclear or cytoplasmic RNA. This assay can, optionally, be normalized using mRNA transcribed from a control gene, such as a gene encoding glyceraldehyde phosphate dehydrogenase (GAPDH). Levels of nuclear and cytoplasmic RNA accumulation can then be compared for novel DNAs of the invention to determine whether an increase has occurred following correction of one or more consensus or near consensus splice sites, and/or by addition of one or more non-naturally occurring introns into the DNA.  
     [0120] Novel DNAs of the invention can also be assayed for altered splicing patterns using similar techniques. For example, as described in the examples below, to determine whether a non-naturally occurring intron has been successfully incorporated into a DNA so that it is correctly spliced during mRNA processing, cytoplasmic mRNA can be assayed by Northern blot analysis, reverse transcriptase PCR (RT-PCR), or RNase protection assays. Such assays are used to determine the size of the mRNA produced from the novel DNA containing the non-naturally occurring intron. The size of the mRNA can then be compared to the size of the DNA with and without the intron to determine whether splicing has been achieved, and whether the splicing pattern corresponds to that expected based on the size of the added intron.  
     [0121] Alternatively, protein expressed from cytoplasmic RNA can be assayed by SDS-PAGE analysis and sequenced to confirm that correct splicing has been achieved.  
     [0122] To measure expression levels, novel DNAs of the invention can also be tested in a variety of art-recognized expression assays. Suitable expression assays, as illustrated in the examples provided below, include quantitative ELISA (Zatloukal et al. (1994)  PNAS  91:5148-5152), radioimmunoassay (RIA), and enzyme activity assays. When expression of Factor VIII protein is being measured, in particular, Factor VIII activity assays such as the KabiCoATest, (Kabi Inc., Sweden) can be employed to quantify expression.  
     [0123] Gene Delivery to Cells  
     [0124] Following insertion into an appropriate vector, novel DNAs of the invention can be delivered to cells either in vitro or in vivo. For example, the DNA can be transfected into cells in vitro using standard transfection techniques, such as calcium phosphate precipitation (O&#39;Mahoney et al. (1994)  DNA &amp; Cell Biol.  13(12): 1227-1232). Alternatively, the gene can be delivered to cells in vivo by, for example, intravenous or intramuscular injection.  
     [0125] In one embodiment of the invention, the gene is targeted for delivery to a specific cell by linking the plasmid to a carrier molecule containing a ligand which binds to a component on the surface of a cell, thereby forming a polynucleotide-carrier complex. The carrier can further comprise a nucleic acid binding agent which noncovalently mediates linkage of the DNA to the ligand of the carrier molecule.  
     [0126] The carrier molecule of the polynucleotide-carrier complex performs at least two functions: (1) it binds the polynucleotide (e.g., the plasmid) in a manner which is sufficiently stable (either in vivo, ex vivo, or in vitro) to prevent significant uncoupling of the polynucleotide extracellularly prior to internalization by a target cell, and (2) it binds to a component on the surface of a target cell so that the polynucleotide-carrier complex is internalized by the cell. Generally, the carrier is made up of a cell-specific ligand and a cationic moiety which, for example are conjugated. The cell-specific ligand binds to a cell surface component, such as a protein, polypeptide, carbohydrate, lipid or combination thereof. It typically binds to a cell surface receptor. The cationic moiety binds, e.g., electrostatically, to the polynucleotide.  
     [0127] The ligand of the carrier molecule can be any natural or synthetic ligand which binds a cell surface receptor. The ligand can be a protein, polypeptide, glycoprotein, glycopeptide, glycolipid or synthetic carbohydrate which has functional groups that are exposed sufficiently to be recognized by the cell surface component. It can also be a component of a biological organism such as a virus, cells (e.g., mammalian, bacterial, protozoan).  
     [0128] Alternatively, the ligand can comprise an antibody, antibody fragment (e.g., an F(ab′) 2  fragment) or analogues thereof (e.g., single chain antibodies) which binds the cell surface component (see e.g., Chen et al. (1994)  FEBS Letters  338:167-169, Ferkol et al. (1993)  J. Clin. Invest.  92:2394-2400, and Rojanasakul et al. (1994)  Pharmaceutical Res.  11(12):1731-1736). Such antibodies can be produced by standard procedures.  
     [0129] Ligands useful in forming the carrier will vary according to the particular cell to be targeted. For targeting hepatocytes, proteins, polypeptides and synthetic compounds containing galactose-terminal carbohydrates, such as carbohydrate trees obtained from natural glycoproteins or chemically synthesized, can be used. For example, natural glycoproteins that either contain terminal galactose residues or can be enzymatically treated to expose terminal galactose residues (e.g., by chemical or enzymatic desialylation) can be used. In one embodiment, the ligand is an asialoglycoprotein, such as asialoorosomucoid, asialofetuin or desialylated vesicular stomatitis virus. In another embodiment, the ligand is a tri- or tetra-antennary carbohydrate moiety.  
     [0130] Alternatively, suitable ligands for targeting hepatocytes can be prepared by chemically coupling galactose-terminal carbohydrates (e.g., galactose, mannose, lactose, arabinogalactan etc.) to nongalactose-bearing proteins or polypeptides (e.g., polycations) by, for example, reductive lactosamination. Methods of forming a broad variety of other synthetic glycoproteins having exposed terminal galactose residues, all of which can be used to target hepatocytes, are described, for example, by Chen et al. (1994)  Human Gene Therapy  5:429-435 and Ferkol et al. (1993)  FASEB  7: 1081-1091 (galactosylation of polycationic histones and albumins using EDC); Perales et al. (1994)  PNAS  91:4086-4090 and Midoux et al. (1993)  Nucleic Acids Research  21(4):871-878 (lactosylation and galactosylation of polylysine using α-D-galactopyranosyl phenylisothiocyanate and 4-isothiocyanatophenyl β-D-lactoside); Martinez-Fong (1994)  Hepatology  20(6):1602-1608 (lactosylation of polylysine using sodium cyanoborohydride and preparation of asialofetuin-polylysine conjugates using SPDP); and Plank et al. (1992)  Bioconjugate Chem.  3:533-539 (reductive coupling of four terminal galactose residues to a synthetic carrier peptide, followed by linking the carrier to polylysine using SPDP).  
     [0131] For targeting the polynucleotide-carrier complex to other cell surface receptors, the carrier component of the complex can comprise other types of ligands. For example, mannose can be used to target macrophages (lymphoma) and Kupffer cells, mannose 6-phosphate glycoproteins can be used to target fibroblasts (fibro-sarcoma), intrinsic factor-vitamin B12 and bile acids (See Kramer et al. (1992)  J. Biol. Chem.  267:18598-18604) can be used to target enterocytes, insulin can be used to target fat cells and muscle cells (see e.g., Rosenkranz et al. (1992)  Experimental Cell Research  199:323-329 and Huckett et al. (1990)  Chemical Pharmacology  40(2):253-263), transferrin can be used to target smooth muscle cells (see e.g., Wagner et al. (1990)  PNAS  87:3410-3414 and U.S. Pat. No. 5, 354,844 (Beug et al.)), Apolipoprotein E can be used to target nerve cells, and pulmonary surfactants, such as Protein A, can be used to target epithelial cells (see e.g., Ross et al. (1995)  Human Gene Therapy  6:31-40).  
     [0132] The cationic moiety of the carrier molecule can be any positively charged species capable of electrostatically binding to negatively charged polynucleotides. Preferred cationic moieties for use in the carrier are polycations, such as polylysine (e.g., poly-L-lysine), polyarginine, polyornithine, spermine, basic proteins such as histones (Chen et al., supra.), avidin, protamines (see e.g., Wagner et al., supra.), modified albumin (i.e., N-acylurea albumin) (see e.g., Huckett et al., supra.) and polyamidoamine cascade polymers (see e.g., Haensler et al. (1993)  Bioconjugate Chem.  4: 372-379). A preferred polycation is polylysine (e.g., ranging from 3,800 to 60,000 daltons). Other preferred cationic moieties for use in the carrier are cationic liposomes.  
     [0133] In one embodiment, the carrier comprises polylysine having a molecular weight of about 17,000 daltons (purchased as the hydrogen bromide salt having a MW of a 26,000 daltons), corresponding to a chain length of approximately 100-120 lysine residues. In another embodiment, the carrier comprises a polycation having a molecular weight of about 2,600 daltons (purchased as the hydrogen bromide salt having a MW of a 4,000 daltons), corresponding to a chain length of approximately 15-10 lysine residues.  
     [0134] The carrier can be formed by linking a cationic moiety and a cell-specific ligand using standard cross-linking reagents which are well known in the art. The linkage is typically covalent. A preferred linkage is a peptide bond. This can be formed with a water soluble carbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), as described by McKee et al (1994)  Bioconjugate Chem.  5: 306-311 or Jung, G. et al. (1981)  Biochem. Biophys. Res. Commun.  101: 599-606 or Grabarek et al. (1990)  Anal. Biochem.  185:131. Alternative linkages are disulfide bonds which can be formed using cross-linking reagents, such as N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-hydroxysuccinimidyl ester of chlorambucil, N-Succinimidyl-(4-Iodoacetyl)aminobenzoate) (SIAB), Sulfo-SIAB, and Sulfo-succinimidyl-4-maleimidophenyl-butyrate (Sulfo-SMPB). Strong noncovalent linkages, such as avidin-biotin interactions, can also be used to link cationic moieties to a variety of cell binding agents to form suitable carrier molecules.  
     [0135] The linkage reaction can be optimized for the particular cationic moiety and cell binding agent used to form the carrier. The optimal ratio (w:w) of cationic moiety to cell binding agent can be determined empirically. This ratio will vary with the size of the cationic moiety (e.g., polycation) being used in the carrier, and with the size of the polynucleotide to be complexed. However, this ratio generally ranges from about 0.2-5.0 (cationic moiety ligand). Uncoupled components and aggregates can be separated from the carrier by molecular sieve or ion exchange chromatography (e.g., Aquapore™ cation exchange, Rainin).  
     [0136] In one embodiment of the invention, a carrier made up of a conjugate of asialoorosomucoid and polylysine is formed with the cross linking agent 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide. After dialysis, the conjugate can be separated from unconjugated components by preparative acid-urea polyacrylamide gel electrophoresis (pH 4-5).  
     [0137] Following formation of the carrier molecule, the polynucleotide (e.g., plasmid) is linked to the carrier so that (a) the polynucleotide is sufficiently stable (either in vivo, ex vivo, or in vitro) to prevent significant uncoupling of the polynucleotide extracellularly prior to internalization by the target cell, (b) the polynucleotide is released in functional form under appropriate conditions within the cell, (c) the polynucleotide is not damaged and (d) the carrier retains its capacity to bind to cells. Generally, the linkage between the carrier and the polynucleotide is noncovalent. Appropriate noncovalent bonds include, for example, electrostatic bonds, hydrogen bonds, hydrophobic bonds, anti-polynucleotide antibody binding, linkages mediated by intercalating agents, and streptavidin or avidin binding to polynucleotide-containing biotinylated nucleotides. However, the carrier can also be directly (e.g., covalently) linked to the polynucleotide using, for example, chemical cross-linking agents (e.g., as described in WO-A-91/04753 (Cetus Corp.), entitled “Conjugates of Antisense Oligonucleotides and Therapeutic Uses Thereof”).  
     [0138] As described in Example 4, polynucleotide-carrier complexes can be formed by combining a solution containing carrier molecules with a solution containing a polynucleotide to be complexed, preferably so that the resulting composition is isotonic (see Example 4).  
     [0139] Administration  
     [0140] Novel DNAs of the invention can be administered to cells either in vitro or in vivo for transcription and/or expression therein.  
     [0141] For in vitro delivery, cultured cells can be incubated with the DNA in an appropriate medium under suitable transfection conditions, as is well known in the art.  
     [0142] For in vivo delivery (e.g., in methods of gene therapy) DNAs of the invention (preferably contained within a suitable expression vector) can be administered to a subject in a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier”, as used herein, is intended to include any physiologically acceptable vehicle for stabilizing DNAs of the present invention for administration in vivo, including, for example, saline and aqueous buffer solutions, solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media is incompatible with the polynucleotide-carrier complexes of the present invention, use thereof in a therapeutic composition is contemplated.  
     [0143] Accordingly, novel DNAs of the invention can be combined with pharmaceutically acceptable carriers to form a pharmaceutical composition. In all cases, the pharmaceutical composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action or microorganisms such as bacteria and fungi. Protection of the polynucleotide-carrier complexes from degradative enzymes (e.g., nucleases) can be achieved by including in the composition a protective coating or nuclease inhibitor. Prevention of the action of microorganisms can be achieved by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.  
     [0144] Novel DNAs of the invention may be administered in vivo by any suitable route of administration. The appropriate dosage may vary according to the selected route of administration. The DNAs are preferably injected intravenously in solution containing a pharmaceutically acceptable carrier, as defined herein. Sterile injectable solutions can be prepared by incorporating the DNA in the required amount in an appropriate buffer with one or a combination of ingredients enumerated above or below, followed by filtered sterilization. Other suitable routes of administration include intravascular, subcutaneous (including slow-release implants), topical and oral.  
     [0145] Appropriate dosages may be determined empirically, as is routinely practiced in the art. For example, mice can be administered dosages of up to 1.0 mg of DNA per 20 g of mouse, or about 1.0 mL of DNA in solution per 1.4 mL of mouse blood.  
     [0146] Administration of a novel DNA, or protein expressed therefrom, to a subject can be in any pharmacological form including a therapeutically active amount of DNA or protein, in combination with another therapeutic molecule. Administration of a therapeutically active amount of a pharmaceutical composition of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result (e.g., an improvement in clinical symptoms). A therapeutically active amount of DNA or protein may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.  
     [0147] Uses  
     [0148] Novel DNAs of the present invention can be used to efficiently express a desired protein within a cell. Accordingly, such DNAs can be used in any context in which gene transcription and/or expression is desired.  
     [0149] In one embodiment, the DNA is used in a method of gene therapy to treat a clinical disorder. In another embodiment, the DNA is used in antisense therapy to produce sufficient levels of nuclear and/or cytoplasmic mRNA to inhibit expression of a gene. In another embodiment, the DNA is used to study RNA processing and/or gene regulation in vitro or in vivo. In another embodiment, the DNA is used to produce therapeutic or diagnostic proteins which can then be administered to patients as exogenous proteins.  
     [0150] Methods for increasing levels of cytoplasmic RNA accumulation and gene expression provided by the present invention can also be used for any and all of the foregoing purposes.  
     [0151] In a preferred embodiment, the invention provides a method if increasing expression of a gene encoding human Factor VIII. Accordingly, the invention also provides an improved method of human Factor VIII gene therapy involving administering to a patient afflicted with a disease characterized by a deficiency in Factor VIII a novel Factor VIII gene in an amount sufficient to treat the disease.  
     [0152] In addition, the present invention provides a novel method for altering the transcription pattern of a DNA. By correcting one or more consensus or near consensus splice sites within the DNA, or by adding one or more introns to the DNA, the natural splicing pattern of the DNA will be modified and, at the same time, expression may be increased. Accordingly, methods of the invention can be used to tailor the transcription of a DNA so that a greater amount of a particular desired RNA species is transcribed and ultimately expressed, relative to other RNA species transcribed from the DNA (i.e., alternatively spliced RNAs).  
     [0153] Methods of the invention can also be used to modify the coding sequence of a given DNA, so that the structure of the protein expressed from the DNA is altered in a beneficial manner. For example, introns can be added to the DNA so that portions of the gene will be removed during transcription and, thus, not be expressed. Preferred gene portions for removal in this manner include those encoding, e.g., antigenic regions of a protein and/or regions not required for activity. Alternatively or additionally, consensus or near consensus splice sites can be corrected within the DNA so that previously recognizable (i.e., operable) introns and exons are no longer recognized by a cells splicing machinery. This alters the coding sequence of the mRNA ultimately transcribed from the DNA, and can also facilitate its export from the nucleus to the cytoplasm where it can be expressed.  
     [0154] This invention is illustrated further by the following examples which should not be construed as further limiting the subject invention. The contents of all references and published patent applications cited throughout this application are hereby incorporated by reference.  
     EXAMPLES  
     Example 1  
     Construction of a Human Factor VIII Gene Containing an Intron Spanning the β-Domain  
     [0155] A full-length human Factor VIII cDNA containing an intron spanning the section of the cDNA encoding amino acids 745-1638 (FIG. 11) was constructed as described below. Amino acid numbering was designated starting with Met-1 of the mature human Factor VIII protein and, thus, does not include the 19 amino acid signal peptide of the protein. The β-domain region of a human Factor VIII protein is made up of 983 amino acids (Vehar et al. (1984)  Nature  312: 337-342). Thus, the region of the cDNA spliced out during pre-mRNA processing corresponds to about 89% of the β-domain.  
     [0156] To select suitable sites for inserting the 5′ splice donor (SD) and 3′ splice acceptor (SA) sites, the sequence of the full-length Factor VIII cDNA expression plasmid pCY-6 (SEQ ID NO:4) was scanned for convenient restriction enzyme sites. Restriction sites were selected according to the following criteria: (a) they flanked and were in close proximity to the sites into which the splicing signals were to be introduced, so that any PCR fragment generated to fill in the region between these sites would have as little chance as possible for undesired point mutations introduced by the process of PCR; (b) they would cut the expression plasmid in as few places as possible, preferably only at the site flanking the region of splice site introduction.  
     [0157] The restriction sites chosen according to these criteria for cloning in the splice donor site were: Kpn I (base 2816 of the coding sequence of pCY-6., or base 3822 of the complete nucleotide sequence of pCY-6 provided in SEQ ID NO:4, since the first 1005 bases of this plasmid are non-coding bases), and Tth 1111 (base 3449 of the coding sequence of pCY-6, or base 4455 of the complete nucleotide sequence of pCY-6 shown in SEQ ID NO:4). The restriction sites chosen according to these criteria for cloning in the splice acceptor site were: Bcl I (bases 1407 and 5424 of the coding sequence of pCY-6, or bases 2413 and 6430 of the complete nucleotide sequence of pCY-6 shown in SEQ ID NO:4) and BspE 1 (base 7228 of the coding sequence of pCY-6, or base 8234 of the complete nucleotide sequence of pCY-6 shown in SEQ ID NO:4).  
     [0158] Generation of Splice Donor Site  
     [0159] A fragment containing the region of Factor VIII cDNA from the Kpn I site to the Tth 111 I site, with the above described splice donor sequence inserted at the appropriate spot, was then generated in the following manner:  
     [0160] A. PCR primers were designed, such that the top strand upstream primer (Fragment A top) would prime at the Kpn I site of full-length Factor VIII cDNA (FIG. 12), and the bottom strand downstream primer (Fragment A bottom) would prime at the site of insertion for the 5′ splice donor. The bottom strand primer also contained the insertion sequence. These primers were used in a PCR reaction with pCIS-F8 (full-length Factor VIII cDNA expression plasmid) as template to yield “Fragment A.” which contains the sequence spanning the region of Factor VIII cDNA from Kpn I to the splice donor insertion site, located at the 3′ end of the fragment.  
     [0161] B. In similar fashion, “Fragment B” was generated using primer “Fragment B top,” which contains the insertion sequence, and would prime at the insertion site of full-length Factor VIII cDNA, and primer “Fragment B bottom,” which would prime at the Tth 111 I site of full-length Factor VIII cDNA. “Fragment B” contains the sequence spanning the region of Factor VIII cDNA from the splice donor insertion site to Tth 111I. The 5′ splice donor insertion sequence was located at the 5′ end of the fragment.  
     [0162] C. Fragments A and B were run on a horizontal agarose gel, excised, and extracted, in order to purify them away from unincorporated nucleotides and primers.  
     [0163] D. These fragments were then combined in a PCR reaction using as primers “Fragment A top” and “Fragment B bottom.” The regions at the 3′ end of Fragment A and the 5′ end of Fragment B overlapped because they were identical, and the final product of this reaction was a PCR fragment spanning the Factor VIII cDNA from Kpn I to Tth 111I, and containing the engineered splice donor at the insertion site, i.e., near the beginning of the coding region of the β-domain of Factor VIII. This fragment was designated “Fragment AB.” 
     [0164] E. Fragment AB (an overlap PCR product) was cloned into the EcoR V site of pBluescript II SK(+) to yield clone pBS-SD (FIG. 9), and the sequence of the insertion was then confirmed.  
     [0165] Generation of Splice Acceptor Site  
     [0166] A fragment containing the region of Factor VIII cDNA from the second Bcl I site to the BspE I site, with the above described splice acceptor sequence inserted at the appropriate spot, was generated in the following manner:  
     [0167] A. PCR primers were designed, such that the top strand upstream primer (Primer A) would prime at the second Bcl I site, and the bottom strand downstream primer (Primer B2) would prime at the insertion site for the 3′ splice acceptor. The bottom strand primer also contained the restriction sites Mun I and BspE I. These primers were used in a PCR reaction with pCIS-F8 as template to yield “Fragment I,” which contains the sequence spanning the region of Factor VIII cDNA from the Bcl I site to the insertion site, with the Mun I and BspE I sites located at the 3′ end of the fragment.  
     [0168] B. In a similar fashion, “Fragment III” was generated using “Primer G3” which contains the restriction site BstE II, the splice acceptor recognition sequence (polypyrimidine tract followed by “CAG”), and primes at the insertion site for the splice acceptor; and “Primer H,” which would prime the bottom strand at the BspE I site, so that the resulting fragment would contain the restriction site BstE II, the splice acceptor recognition site and sequence spanning the region of Factor VIII cDNA from the insertion site to BspE I.  
     [0169] C. “Fragment II,” which contained the branch signals and IVS 14 sequence, was generated by designing four oligos (C2, D, E, and F3), two top and two bottom, which, when combined, would overlap each other by 21 to 22 bases, and when filled in and amplified under PCR conditions, would generate a fragment containing a Mun I site, 130 bases of the aforementioned IVS 14 sequence (including the 2 branch sequences at the 5′ end of the 130 bases), and the cloning sites BstE II and BspE I. In addition, two small primers (CX and FX2) were designed that would prime at the very ends of the expected fragment, in order to increase amplification of full-length PCR product. All oligonucleotide primers were combined in a single PCR reaction, and the desired fragment was generated.  
     [0170] D. All three fragments were cloned into the EcoR V site of pBluescript II SK(+), and their sequences were then confirmed.  
     [0171] E. Fragment II was isolated out of pBluescript as a Mun 1 to BspE I fragment, and cloned into the pBluescript-Fragment I clone at the corresponding sites, to yield clone pBS-FI/FII (FIG. 9), Fragment III was isolated out of pBluescript as a BstE II to BspE I fragment, and cloned into the corresponding sites of pBS-FI/FII to yield pBS-FI/FII/FIII (FIG. 9). This final bluescript clone contained the region spanning Factor VIII cDNA from the second Bcl I site to the BspE I site, and contained the IVS 14 and splice acceptor sequence inserted at the appropriate sites. The pBS-FI/FII/FIII clone was then sequenced.  
     [0172] Cloning Splice Donor and Acceptor Sites into a Factor VIII cDNA Vector (pCY-6)  
     [0173] Fragment AB and Fragment I/II/III were isolated out of pBluescript and cloned into pCY-6 in the following manner:  
     [0174] A. Fragment I/II/III was isolated from pBS-FI/FII/FIII as a Bcl I to BspE I fragment.  
     [0175] B. pCY-601 was digested to completion with BspE I, linearizing the plasmid. This linear DNA was partially digested with Bcl I for 5 minutes, and then immediately run on a gel. The band corresponding to a fragment which had been cut only at the BspE I and the second Bcl I site was isolated and extracted from the agarose gel. This isolated fragment was ligated to Fragment I/II/III and yielded pCY-601/FI/FII/FIII (FIG. 9).  
     [0176] C. Fragment AB was isolated from pBS-SD as a Kpn I to Tth111 I fragment, and cloned into the corresponding sites of pCY-601FI/FII/FIII to yield pLZ-601.  
     [0177] D. Plasmids pCY-6 and pLZ-601 were digested sequentially with enzymes Nco I and Sal I. The small fragment of the pCY-6 digest and the large fragment of the pLZ-601 digest were isolated and ligated together to yield plasmid pLZ-6, a second β-domain intron Factor VIII expression plasmid.  
     [0178] pCY-6- and pCY-601 are expression plasmids for full-length Factor VIII cDNA. The difference between the two is that the former contains an intron in the 5′ untranslated region of the Factor VIII transcript, derived from the second IVS of rabbit beta globin gene. The latter lacks this engineered IVS. In vitro experiments have shown that pCY-601 yields undetectable levels of Factor VIII, while pCY-6 yields low but detectable Factor VIII levels.  
     [0179] Expression Assays  
     [0180] To test expression of the various Factor VIII cDNA plasmids including those created as described above, plasmids were transfected at a concentration of 2.0-2.5 μg/ml into HuH-7 human carcinoma cells using the calcium phosphate precipitation method described by O&#39;Mahoney et al. (1994)  DNA  &amp;  Cell Biol.  13(12): 1227-1232. Expression levels were measured using the KabiCoATest (Kabi Inc., Sweden). This is both a quantitative and a qualitative assay for measuring Factor VIII expression, because it measures enzymatic activity of Factor VIII.  
     [0181] Reverse Transcriptase-PCR Analysis of Cells Transfected With Factor VIII Expression Plasmids  
     [0182] To confirm that the engineered intron spanning the β-domain of the Factor VIII cDNA in plasmid pLZ-6 resulted in proper splicing of the β-domain coding region, reverse transcriptase (RT)-PCR analysis was performed as follows:  
     [0183] HUH7 cells in T-75 flasks were transfected via CaPO 4  precipitation with 36 μg of each of the following DNA plasmids:  
     [0184] pCY-2 β-domain deleted human Factor VIII cDNA  
     [0185] pCY-6 Full-length human Factor VIII cDNA  
     [0186] pLZ-6 Full length human Factor VIII cDNA with engineered β-domain intron  
     [0187] 75 ng of pCMVhGH was co-transfected as a transfection control. Untransfected cells were grown alongside as a negative control.  
     [0188] Total RNA was isolated from cells 24 hours post-transfection using Gibco BRL Trizol reagent, according to the standard protocol included in product insert.  
     [0189] RT-PCR Experiments were performed as follows: RT-PCR was performed on all RNA preps to characterize RNA. “Minus RT” PCR was performed on all RNA preps as a negative control (without RT, only DNA is amplified). PCR was performed on plasmids used in transfection assays to compare with RT-PCRs of the RNA preps. All RT-PCR was performed with Access RT-PCR system (Promega, Cat. #A1250). In each 50 μl reaction, 1.0 μg total RNA was used as template. Primer pairs were designed according to Factor VIII sequences as follows: the 5′ primer anneals to the top strand of Factor VIII, about 250 base pairs upstream of the β-domain junction; while the 3′ primer anneals to the bottom strand of Factor VIII, about 250 base pairs downstream of the β-domain junction.  
     [0190] The nucleotide sequences of the primers used to characterize (i.e., confirm) the β-domain intron splicing were as follows:  
                                          5′ primer                   TS 2921-2940:     5′ TGG TCT ATG AAG ACA CAC TC 3′             (20 mer)                       3′ primer           BS 6261-6280:     5′ TGA GCC CTG TTT CTT AGA AC 3′             (20 mer)          
 
     [0191] RT-PCR files were set up according to manufacturer&#39;s recommendation:  
     [0192] 48° C., 45 minutes; ×1 cycle  
     [0193] 94° C., 2 minutes; ×1 cycle  
     [0194] 94° C., 30 sec; ×40 cycles  
     [0195] 60° C., 1 min; ×40 cycles  
     [0196] 68° C., 2 min; ×40 cycles  
     [0197] 68° C., 7 min; ×1 cycle  
     [0198] 4° C., soak overnight  
     [0199] The data obtained from the RT-PCR assays demonstrated that engineered β-domain intron was spliced as predicted. The RT-PCR product (˜500 bp) generated from pLZ-6 (containing the β-domain intron) was similar to that obtained from pCY-2 (containing β-domain deleted Factor VIII cDNA). The RT-PCR product observed for pCY-6 (containing the full length Factor VIII cDNA) yielded a much larger band (˜3.3 kb).  
     [0200] In the control groups, it was confirmed that DNA from the Huh-7 cells transfected with various Factor VIII constructs were consistent with regular PCR results of the corresponding plasmids. Background bands from untransfected Huh-7 cells were presumably contributed by cross-over during sample handling. This can be further investigated by using polyA +  RNA as template, as well as by setting up RT-PCR with different primer sets.  
     Example 2  
     Correction of Consensus and near Consensus Splice Sites Within a Human Factor VIII Gene  
     [0201] Plasmid pCY-2, containing the coding region of the β-domain deleted human Factor VIII cDNA (nucleotides 1006-5379 of SEQ ID NO:2), was analyzed using the MacVector™ program for consensus and near consensus (a) splice donor sites, (b) splice acceptor sites and (c) branch sequences. Near consensus 5′ splice donor sites were selected using the following criteria: sites were required to contain at least 5 out of the 9 splice donor consensus bases (i.e., (C/A)AG GT (A/G)AGT), including the invariant  GT , provided that if only 5 out of 9 bases were present, these 5 bases were located consecutively in a row. Near consensus 3′ splice acceptor sites were selected using the following criteria: sites were required to contain at least 3 out of the following 14 splice acceptor consensus bases (Y=10)C AG G (wherein Y is a pyrimidine within the pyrimidine track), including the invariant  AG . Only branch sequences which were 100% consensus were searched for.  
     [0202] Using these criteria, 23 near consensus 5′ splice donor sequences, 22 near consensus 3′ splice acceptor sequences, and 18 consensus branch sequences were identified. No consensus 5′ splice donor or 3′ splice acceptor sequences were identified. To correct these near consensus splice donor and acceptor sequences, and consensus branch sequences, it was first determined whether the invariant  GT ,  AG , or  A  bases within the site could be substituted without changing the coding sequence of the site. If they could be, then these conservative (silent) substitutions were made, thereby rendering the site non-consensus (since the invariant bases are required for recognition as a splice site).  
     [0203] If the invariant bases within selected consensus and near consensus sites could not be substituted without changing the coding sequence of the site (i.e., if no degeneracy existed for the amino acid sequence coded for), then the maximum number of silent point mutations were made to render the site as far from consensus as possible. All bases which contributed to homology of the consensus or near consensus site with the corresponding consensus sequence, and which were able to be conservatively substituted (with non-consensus bases), were mutated.  
     [0204] Using these guidelines, 99 silent point mutations were selected, as shown in FIGS.  4 A- 4 C. The positions of each of these silent point mutations is shown in FIG. 3.  
     [0205] To prepare a new pCY-2 human β-domain deleted Factor VIII cDNA coding sequence which contains the above-described corrections, the following procedure can be used:  
     [0206] Overlapping 60-mer oligonucleotides can be synthesized based on the coding sequence of pCY2. Each of the 185 oligonucleotide contains the desired corrections. These oligonucleotides are then assembled in five segments (shown in FIG. 9) using the method of Stemmer et al. (1995)  Gene  164: 49-53. Prior to assembly, each segment can be sequenced and tested in in vitro transfection assays (nuclear and cytoplasmic RNA analysis) in pCY2. A schematic illustration of this process is shown in FIG. 8. The plasmid containing the new corrected coding sequence is desginated “pDJC.” 
     [0207] To test expression levels of pDJC, the plasmid can be transfected at a concentration of 2.0-2.5 μg/ml into HuH-7 human carcinoma cells using any suitable transfection technique, such as the calcium phosphate precipitation method described by O&#39;Mahoney et al. (1994)  DNA  &amp;  Cell Biol.  13(12): 1227-1232. Factor VIII expression can then be measured using the KabiCoATest (Kabi Inc., Sweden). This is both a quantitative and a qualitative assay for measuring Factor VIII expression, because it measures enzymatic activity of Factor VIII. Alternatively, plasmids such as pDJC can be tested for in vivo expression using the procedure described below in Example 4.  
     Example 3  
     Optimized Expression Vectors  
     [0208] Optimized expression vectors for liver-specific and endothelium-specific human Factor VIII expression were prepared and tested as follows:  
     [0209] The β-domain deleted human Factor VIII cDNA was obtained through Bayer Corporation in plasmid p25D, having a coding sequence corresponding to nucleotides 1006-5379 of SEQ ID NO:2. The human thyroid binding globulin promoter (TBG) (bases −382 to +3) was obtained by PCR from human liver genomic DNA (Hayashi et al. (1993) Mol. Endo. 7: 1049). The human endothelin-1 (ET-1) gene promoter (Lee et al. (1990)  J. Biol. Chem.  265(18) was synthesized by amplification of overlapping oligos in a PCR reaction.  
     [0210] After sequence confirmation, the TBG and ET-1 promoters were cloned into two separate vectors upstream of an optimized leader sequence (SEQ ID NO:11), using standard cloning techniques. The leader sequence was designed in a similar manner to that reported by Kozak et al. (1994)  J. Mol. Biol.  235:95) and synthesized (Retrogen Inc., San Diego, Calif.) as 71 base pair top and bottom strand oligos, annealed and cloned upstream of the Factor VIII ATG. The 126 base pair intron-1 of the rabbit β-globin gene, containing the nucleotide sequence modifications shown in FIG. 23 (SEQ ID NO:7), was also synthesized and inserted into the leader sequence following base 42 of the 71 nucleotide sequence.  
     [0211] In the construct containing the TBG promoter, top and bottom strands of the human alpha-1 microglobulin/bikunin enhancer (ABP), sequences −2804 through −2704 (Rouet et al. (1992)  J. Biol. Chem.  267:20765), were synthesized, annealed and cloned upstream of the promoter. Cloning sites flanking the enhancer were designed to facilitate easy multimerization. In the construct containing the ES-1 promoter, top and bottom strands of the human c-fos SRE enhancer (Treisman et al. (1986)  Cell  46) were synthesized, annealed and cloned upstream of the promoter.  
     [0212] The post-transcriptional regulatory element (PRE) from hepatitis B virus, was isolated from plasmid Adw-HTD as a 587 base-pair Stu I-Stu I fragment. It was cloned into the 3′ UTR of the Factor VIII construct (at the Hpa I site) containing the TBG promoter and ABP enhancers, upstream of the polyadenylation sequence. A two copy PRE element was isolated as a Spe I-Spe I fragment from an early vector where two copies had ligated together. This fragment was converted to a blunt end fragment by the Klenow fragment of  E-coli  DNA polymerase I and also cloned into the Factor VIII construct at the same Hpa I site.  
     [0213] Thus, the following constructs were produced using the foregoing materials and methods:  
     [0214] Plasmid pCY-2 having a 5′ untranslated region containing the TBG promoter, two copies of the ABP enhancer; and the modified rabbit β-globin IVS, all upstream of the human β-domain deleted Factor VIII gene.  
     [0215] Plasmid pCY2-SE5 which was identical to pCY-2, except that the TBG promoter was replaced by the ET-1 gene promoter, and the ABP enhancers (both copies) were replaced by one copy of the SRE enhancer.  
     [0216] Plasmid pCY-201 which was identical to pCY-2, except that it lacked the 5′ intron.  
     [0217] Plasmid pCY-401 and pCY-402 which were identical to pCY-201, except that they contained one and two copies of the HBV PRE, respectively.  
     [0218] Expression levels for each of the foregoing gene constructs was compared in human hepatoma cells (HUH-7) maintained in DMEM (Dulbecco&#39;s modified Eagle medium (GIBCO BRL), supplemented with 10% heat inactivated fetal calf serum (10% FCS), penicillin (50 IU/ml), and streptomycin (50 μg/ml) in a humidified atmosphere of 5% CO 2  at 37° C. For experiments involving quantitation of human factor VIII protein, media was supplemented with an additional 10% FCS. DNA transfection was performed by a calcium phosphate coprecipitation method.  
     [0219] Other human Factor VIII gene constructs (shown below in Table I) tested for expression, prepared as described above, included constructs which were identical to pCY-2, except that they contained (a) the TBG promoter with no enhancer or 5′ intron, (b) the TBG promoter with a 5′ modified rabbit β-globin intron (present within the leader sequence), but no enhancer, (c) the TBG promoter with one copy of the ABP enhancer and a 5′ modified rabbit β-globin intron (present within the leader sequence), and (d) the TBG promoter with two copies of the ABP enhancer and a 5′ modified rabbit β-globin intron (present within the leader sequence).  
     [0220] Active Factor VIII protein was measured from tissue culture supernatants by COAtest VII:c/4 kit assay specific for active Factor VIII protein. Transfection efficiencies were normalized to expression of cotransfected human growth hormone (hGH).  
     [0221] As shown below in Table I, liver-specific human Factor VIII expression is significantly increased by the combined use of the TBG promoter and a 5′ intron within the 5′ UTR of the gene construct. Expression is further increased (over 30 fold) by adding a copy of the ABP enhancer in the same construct. Expression is still further increased (over 60 fold) by using two copies of the ABP enhancer in the same construct. In addition, as shown in FIG. 18, expression is also significantly increased by adding one or more PRE sequences into the 3′ UTR of the gene construct, although, in this experiment, not as much as by adding a 5′ intron within the 5′ UTR.  
                           TABLE I                                       Fold Increase in Factor           5′ Region Tested   VIII Expression In Vitro                                                    TBG Promoter   1           TBG Promoter, 5′ Intron   3.5           ABP Enhancer (1 copy),   30.1           TBG Promoter, 5′ Intron           ABP Enhancer (2 copies),   63.2           TBG Promoter, 5′ Intron           (pCY-2)                      
 
     [0222] Expression of pCY2-SE5 was also tested and compared with pCY-2 in (a) bovine aortic endothelial cells and (b) HUH-7 cells. Transfections and Assays were performed as described above. Significantly more biologically active human Factor VIII was secreted from cells transfected with pCY2-SE5 than with pCY-2 (625 pg/ml vs. 280 pg/ml). While liver-specific pCY-2 expressed more than 10 ng/ml of human Factor VIII from HUH-7 cells, no human Factor VIII could be detected from pCY2-SE5 transfected HUH-7 cells.  
     [0223] Constructs were also tested in vivo. Specifically, pCY-2 and pCY2-SE5 were tested in mouse models by injecting mice (tail vein) with 10 μg of DNA in one 1.0 ml of solution (0.3 M NaCl, pH 9). Plasmids pCY-6, pLZ-6 and pLZ-6A (described in Example 1) were tested in the same experiment. Levels of human Factor VIII were measured in mouse serum. The results are shown in FIG. 19. Plasmid pCY-2, containing the TBG promoter, 2 copies of the ABP enhancer, and an optimized 5′ intron, had the highest expression, followed by pLZ-6A, pLZ-6, pCY2-SE5 and pCY-6.  
     [0224] Plasmid pCY-2 was also tested in vivo in mice, along with plasmid p25D which contained the same coding sequence (for human β-domain deleted Factor VIII) without an optimized 5′ UTR. Specifically, instead of 2 copies of the ABP enhancer, one copy of the TBG promoter and a leader sequence containing an optimized (i.e., modified to contain consensus splice donor and acceptor sites and a consensus branch and pyrimidine track sequence) 5′ rabbit β-globin intron (as contained in the 5′ UTR of pCY-2), p25D contained within its 5′ UTR one copy of the CMV enhancer, one copy of the CMV promoter, and a leader sequence containing an unmodified short (130 bp) chimeric human IgE intron (containing uncorrected near consensus splice donor and acceptor sites). Plasmids were injected into mice (tail vein) in the form of asialoorosomucoid/polylysine/DNA complexes formed as described below in Example 4. Mice were injected with 10 μg of DNA (complexed) in 1.0 of solution (0.3 M NaCl, pH 9).  
     [0225] The results are shown in FIG. 25 and demonstrate that optimization of gene constructs by modification of 5′ UTRs to contain novel combinations of strong tissue-specific promoters and enhancers, and optimized introns (e.g. modified to contain consensus splice donor and acceptor sites and a consensus branch and pyrimidine track sequence) significantly increases both levels and duration of gene expression. Notably, expression of p25D shut off after only 8 days, whereas expression of pCY-2 was maintained at nearly 100% of initial levels (well in the human therapeutic range of 10 ng/ml or more) for over 10 days. In the same experiment, expression was maintained well in the therapeutic range for greater than 30 days.  
     [0226] Overall, the results of the foregoing examples demonstrate that gene expression can be significantly increased and prolonged in vivo by optimizing untranslated regulatory regions and/or coding sequences in accordance with the teachings of the present invention.  
     Example 4  
     Targeted Delivery of Novel Genes to Cells  
     [0227] Novel genes of the invention, such as novel Factor VIII genes contained in appropriate expression vectors, can be selectively delivered to target cells either in vitro or in vivo as follows:  
     [0228] Formation of Targeted Molecular Complexes  
     [0229] I. Reagents  
     [0230] Protamine, poly-L-lysine (4 kD, 10 kD, 26 kD; mean MW) and ethidium bromide can be purchased from Sigma Chemical Co., St. Louis, Mo. 1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide (EDC) can be purchased from Aldrich Chemical Co, Milwaukee, Wis. Synthetic polylysines can be purchased from Research Genetics (Huntsville, Ala.) or Dr. Schwabe (Protein Chemistry Facility at the Medical University of South Carolina). Orosomucoid (OR) can be purchased from Alpha Therapeutics, Los Angeles, Calif. Asialoorosomucoid (AsOR) can be prepared from orosomucoid (15 mg/ml) by hydrolysis with 0.1 N sulfuric acid at 76° C. for one hour. AsOR can then be purified from the reaction mixture by neutralization with 1.0 N NaOH to pH 5.5 and exhaustive dialysis against water at room temperature. AsOR concentration can be determined using an extinction coefficient of 0.92 ml mg −1 , cm −1  at 280 nm. The thiobarbituric acid assay of Warren (1959)  J. Biol. Chem.  234:1971-1975 or of Uchida (1977)  J. Biochem.  82:1425-1433 can be used to verify desialylation of the OR. AsOR prepared by the above method is typically 98% desialyated.  
     [0231] II. Formation of Carrier Molecules  
     [0232] Carrier molecules capable of electrostatically binding to DNA can be prepared as follows: AsOR-poly-L-lysine conjugate (AP26K) can be formed by carbodiimide coupling similar to that reported by McKee (1994)  Bioconj. Chem.  5:306-311. AsOR, 26 kD poly-L-lysine and EDC in a 1:1:0.5 mass ratio can be reacted as follows. EDC (dry) is added directly to a stirring aqueous AsOR solution. Polylysine (26 kD) is then added, the reaction mixture adjusted to pH 5.5-6.0, and stirred for two hours at ambient temperature. The reaction can be quenched by addition of Na 3 PO 4  (200 mM, pH 11) to a final concentration of 10 mM. The AP26K conjugate can be first purified on a Fast Flow Q Sepharose anion exchange chromatography column (Pharmacia) eluted with 50 mM Tris, pH 7.5; and then dialyzed against water.  
     [0233] III. Calculation of Charge Ratios (+/−)  
     [0234] Charge ratios of purified carrier molecules can be determined as follows: Protein-polylysine conjugates (e.g., AsOR-PL or OR-PL) are exhaustively dialyzed against ultra-pure water. An aliquot of the dialyzed conjugate solution is lyophilized, weighed and dissolved in ultra-pure water at a specific concentration (w/v). Since polylysine has minimal absorbance at 280 nm, the AsOR component of AsOR-polylysine (w/v) is calculated using the extinction coefficient at 280 nm. The composition of the conjugate is estimated by comparison of the concentration of the conjugate (w/v) with the concentration of AsOR (w/v) as determined by UV absorbance. The difference between the two determinations can be attributed to the polylysine component of the conjugate. The composition of OR-polylysine can be calculated in the same manner. The ratio of conjugate to DNA (w/w) necessary for specific charge ratios then can be calculated using the determined conjugate composition. Charge ratios for molecular complexes made with, e.g., polylysine or protamine, can be calculated from the amino acid composition.  
     [0235] IV. Complexation with DNA  
     [0236] To form targeted DNA complexes, DNA (e.g., plasmid DNA) is preferably prepared in glycine (e.g., 0.44 M, pH 7), and is then rapidly added to an equal volume of carrier molecule, also in glycine (e.g., 0.44 M, pH 7), so that the final solution is isotonic.  
     [0237] V. Fluorescence Quenching Assay  
     [0238] Binding efficiencies of DNA to various polycationic carrier molecules can be examined using an ethidium bromide-based quenching assay. Solutions can be prepared containing 2.5 μg/ml EtBr and 10 μg/ml DNA (1:5 EtBr:DNA phosphates molar ratio) in a total volume of 1.0 ml. The polycation is added incrementally with fluorescence readings taken at each point using a fluorometer (e.g., a Sequoia-Turner 450), with excitation and emission wavelengths at 540 nm and 585 nm, respectively. Fluorescence readings are preferably adjusted to compensate for the change in volume due to the addition of polycation, if the polycation did not exceed 3% of the original volume. Results can be reported as the percentage of fluorescence relative to that of uncomplexed plasmid DNA (no polycation).  
     [0239] Cell Delivery In Vivo or In Vitro  
     [0240] DNA complexes prepared as described above can be administered in solution to subjects via injection. By way of example, a 0.1-1.0 ml dose of complex in solution can be injected intravenously via the tail vein into adult (e.g., 18-20 gm) BALB/C mice, at a dose ranging from &lt;1.0-10.0 μg of DNA complex per mouse.  
     [0241] Alternatively, DNA complexes can be incubated with cells (e.g., HuH cells) in culture using any suitable transfection protocol known in the art for targeted uptake. Target cells for transfection must contain on their surface a component capable of binding to the cell-binding component of the DNA complex.  
     [0242] Equivalents  
     [0243] Although the invention has been described with reference to its preferred embodiments, other embodiments can achieve the same results. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments described herein. Such equivalents are considered to be within the scope of this invention and are encompassed by the following claims.  
     [0244] Incorporation by Reference  
     [0245] The contents of all references and patents cited herein are hereby incorporated by reference in their entirety.   
    
     
       
         1 
         
           
             
11 
 
           
           
             
               4374 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
             
               CDS  
                1..4374 

 
             
              1 

ATG GAA ATA GAG CTC TCC ACC TGC TTC TTT CTG TGC CTT TTG CGA TTC       48 
Met Glu Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe 
  1               5                  10                  15 

TGC TTT AGT GCC ACC AGA AGA TAC TAC CTG GGT GCA GTG GAA CTG TCA       96 
Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 
             20                  25                  30 

TGG GAC TAT ATG CAA AGT GAT CTC GGA GAG CTG CCT GTG GAC GCA AGA      144 
Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 
         35                  40                  45 

TTT CCT CCT CGC GTG CCA AAA TCT TTT CCA TTC AAC ACC TCA GTC GTG      192 
Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 
     50                  55                  60 

TAC AAA AAG ACT CTG TTT GTA GAA TTC ACG GTT CAC CTT TTC AAC ATC      240 
Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile 
 65                  70                  75                  80 

GCT AAG CCA AGG CCA CCC TGG ATG GGT CTG CTA GGT CCT ACC ATC CAA      288 
Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 
                 85                  90                  95 

GCT GAG GTT TAT GAT ACA GTG GTC ATT ACA CTT AAG AAC ATG GCT TCC      336 
Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 
            100                 105                 110 

CAT CCT GTC TCC CTT CAT GCT GTT GGT GTA TCC TAC TGG AAA GCT TCT      384 
His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 
        115                 120                 125 

GAG GGA GCT GAA TAT GAT GAT CAG ACC AGT CAA AGG GAG AAA GAA GAT      432 
Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 
    130                 135                 140 

GAT AAA GTC TTC CCT GGT GGA AGC CAT ACA TAT GTC TGG CAA GTC CTG      480 
Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu 
145                 150                 155                 160 

AAA GAG AAT GGT CCA ATG GCC TCC GAC CCA CTG TGC CTT ACC TAC TCA      528 
Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 
                165                 170                 175 

TAT CTT TCT CAT GTG GAC CTG GTT AAA GAC TTG AAT TCA GGC CTC ATT      576 
Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 
            180                 185                 190 

GGA GCC CTA CTA GTA TGT AGA GAA GGG AGT CTG GCC AAG GAA AAG ACA      624 
Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 
        195                 200                 205 

CAG ACC TTG CAC AAA TTT ATA CTA CTT TTT GCT GTA TTT GAT GAA GGG      672 
Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 
    210                 215                 220 

AAA AGT TGG CAC TCA GAA ACA AAG AAC TCC CTC ATG CAA GAT AGG GAT      720 
Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp 
225                 230                 235                 240 

GCT GCA TCT GCT CGG GCC TGG CCT AAA ATG CAC ACA GTC AAT GGT TAT      768 
Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 
                245                 250                 255 

GTA AAC AGG AGC CTG CCA GGA CTG ATT GGA TGC CAC AGG AAA TCA GTC      816 
Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 
            260                 265                 270 

TAT TGG CAT GTT ATA GGA ATG GGC ACC ACT CCT GAA GTG CAC TCA ATA      864 
Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 
        275                 280                 285 

TTC CTC GAA GGA CAC ACA TTT CTT GTT AGA AAC CAT CGC CAG GCG TCC      912 
Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 
    290                 295                 300 

TTG GAA ATC TCG CCA ATA ACT TTC CTT ACT GCT CAA ACA CTC CTC ATG      960 
Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met 
305                 310                 315                 320 

GAC CTT GGA CAG TTT CTA CTG TTT TGT CAT ATC TCT TCC CAC CAA CAT     1008 
Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 
                325                 330                 335 

GAT GGC ATG GAA GCT TAT GTC AAA GTA GAC AGC TGT CCA GAG GAA CCC     1056 
Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 
            340                 345                 350 

CAA CTA CGA ATG AAA AAT AAT GAA GAA GCG GAA GAC TAT GAT GAT GAT     1104 
Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 
        355                 360                 365 

CTT ACC GAT TCT GAA ATG GAT GTG GTC AGA TTT GAT GAT GAC AAC TCT     1152 
Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 
    370                 375                 380 

CCT TCC TTT ATC CAA ATT CGC TCA GTT GCC AAG AAG CAT CCT AAA ACT     1200 
Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 
385                 390                 395                 400 

TGG GTA CAT TAC ATT GCT GCT GAA GAG GAG GAC TGG GAC TAT GCT CCC     1248 
Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 
                405                 410                 415 

TTA GTC CTC GCC CCC GAT GAC AGA AGT TAT AAA AGT CAA TAT TTG AAC     1296 
Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 
            420                 425                 430 

AAT GGC CCT CAG CGG ATT GGA AGG AAG TAC AAA AAA GTC CGA TTT ATG     1344 
Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 
        435                 440                 445 

GCA TAC ACA GAT GAA ACC TTT AAG ACT CGT GAA GCT ATT CAG CAT GAA     1392 
Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 
    450                 455                 460 

TCA GGA ATC TTG GGA CCT TTA CTT TAT GGG GAA GTT GGA GAC ACA CTG     1440 
Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 
465                 470                 475                 480 

CTC ATT ATA TTT AAG AAT CAA GCA AGC AGA CCA TAT AAC ATC TAC CCT     1488 
Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 
                485                 490                 495 

CAC GGA ATC ACC GAT GTC CGT CCT TTG TAT TCA CGC AGA TTA CCA AAA     1536 
His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 
            500                 505                 510 

GGA GTA AAA CAT TTG AAG GAT TTT CCA ATT CTG CCC GGA GAA ATA TTC     1584 
Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 
        515                 520                 525 

AAA TAT AAA TGG ACA GTG ACT GTA GAA GAT GGG CCA ACT AAA TCA GAT     1632 
Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 
    530                 535                 540 

CCT CGG TGC CTG ACC CGC TAT TAC TCT AGT TTC GTC AAT ATG GAG AGA     1680 
Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 
545                 550                 555                 560 

GAT CTA GCT TCA GGA CTC ATT GGC CCT CTC CTC ATC TGC TAC AAA GAA     1728 
Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 
                565                 570                 575 

TCT GTA GAT CAA AGA GGA AAC CAG ATA ATG TCA GAC AAG AGG AAT GTC     1776 
Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 
            580                 585                 590 

ATC CTG TTT TCT GTA TTT GAT GAG AAC CGA AGC TGG TAC CTC ACA GAG     1824 
Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 
        595                 600                 605 

AAT ATA CAA CGC TTT CTC CCC AAT CCC GCT GGA GTG CAG CTT GAG GAT     1872 
Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 
    610                 615                 620 

CCA GAG TTC CAA GCC TCC AAC ATC ATG CAC AGC ATC AAT GGC TAT GTT     1920 
Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 
625                 630                 635                 640 

TTC GAT AGT TTG CAG TTG TCA GTT TGT TTG CAT GAA GTA GCA TAC TGG     1968 
Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 
                645                 650                 655 

TAC ATT CTA AGC ATT GGA GCA CAG ACT GAC TTC CTT TCT GTC TTC TTC     2016 
Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 
            660                 665                 670 

TCT GGA TAT ACC TTC AAA CAC AAA ATG GTC TAT GAA GAC ACA CTC ACC     2064 
Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 
        675                 680                 685 

CTA TTC CCA TTC TCC GGA GAA ACT GTC TTC ATG TCG ATG GAA AAC CCA     2112 
Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 
    690                 695                 700 

GGA CTA TGG ATT CTG GGG TGC CAC AAC TCA GAC TTT CGG AAC AGA GGC     2160 
Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 
705                 710                 715                 720 

ATG ACC GCC TTA CTG AAA GTT TCC AGT TGT GAC AAG AAC ACT GGA GAT     2208 
Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 
                725                 730                 735 

TAT TAC GAG GAC AGT TAT GAA GAT ATT TCA GCA TAC TTG CTG AGT AAA     2256 
Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 
            740                 745                 750 

AAC AAT GCC ATT GAA CCA AGA AGC TTC TCC CAG AAC CCA CCA GTC TTG     2304 
Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 
        755                 760                 765 

AAA CGC CAT CAA CGG GAA ATA ACT CGT ACT ACT CTT CAA TCA GAT CAA     2352 
Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln 
    770                 775                 780 

GAG GAA ATT GAC TAT GAT GAT ACC ATA TCA GTT GAA ATG AAG AAG GAA     2400 
Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu 
785                 790                 795                 800 

GAT TTC GAC ATT TAT GAT GAG GAT GAA AAT CAG AGC CCC CGC AGC TTT     2448 
Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 
                805                 810                 815 

CAA AAG AAA ACA CGA CAC TAT TTT ATT GCT GCA GTG GAG AGG CTC TGG     2496 
Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp 
            820                 825                 830 

GAT TAT GGG ATG AGT AGC TCC CCA CAT GTT CTA AGA AAC AGG GCT CAG     2544 
Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln 
        835                 840                 845 

AGT GGC AGT GTC CCT CAG TTC AAG AAA GTA GTA TTC CAG GAA TTT ACC     2592 
Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 
    850                 855                 860 

GAT GGC TCC TTT ACT CAA CCC TTA TAC CGT GGA GAA CTA AAT GAA CAT     2640 
Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His 
865                 870                 875                 880 

TTG GGA CTC CTG GGG CCA TAT ATA AGA GCA GAA GTT GAA GAT AAT ATC     2688 
Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile 
                885                 890                 895 

ATG GTT ACC TTC AGA AAT CAG GCC TCT CGT CCC TAT TCC TTC TAT TCT     2736 
Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser 
            900                 905                 910 

TCC CTC ATA TCA TAT GAG GAA GAT CAG AGG CAA GGA GCA GAA CCT AGA     2784 
Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 
        915                 920                 925 

AAA AAC TTT GTC AAG CCT AAT GAA ACC AAA ACT TAC TTT TGG AAA GTG     2832 
Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val 
    930                 935                 940 

CAA CAT CAT ATG GCA CCC ACT AAA GAT GAG TTT GAC TGC AAA GCC TGG     2880 
Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp 
945                 950                 955                 960 

GCT TAT TTC TCC GAT GTC GAC CTG GAA AAA GAT GTG CAC TCA GGC CTG     2928 
Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu 
                965                 970                 975 

ATT GGA CCC CTT CTG GTC TGC CAC ACC AAC ACA CTG AAC CCT GCT CAT     2976 
Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 
            980                 985                 990 

GGG AGA CAA GTG ACA GTA CAG GAA TTT GCT CTG TTT TTC ACC ATC TTC     3024 
Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 
        995                 1000                1005 

GAT GAG ACC AAA AGC TGG TAC TTC ACT GAA AAT ATG GAA AGA AAC TGC     3072 
Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys 
    1010                1015                1020 

AGG GCT CCC TGC AAT ATC CAG ATG GAA GAT CCC ACT TTT AAA GAG AAT     3120 
Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn 
1025                1030                1035                1040 

TAT CGC TTC CAT GCA ATC AAT GGC TAC ATA ATG GAT ACA CTA CCT GGC     3168 
Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly 
                1045                1050                1055 

TTA GTA ATG GCT CAG GAT CAA AGG ATT CGA TGG TAT CTG CTC AGC ATG     3216 
Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met 
            1060                1065                1070 

GGC AGC AAT GAA AAC ATC CAT TCT ATT CAT TTC TCC GGA CAT GTG TTC     3264 
Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe 
        1075                1080                1085 

ACT GTA CGA AAA AAA GAG GAG TAT AAA ATG GCA CTG TAC AAT CTC TAT     3312 
Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr 
    1090                1095                1100 

CCC GGA GTT TTC GAG ACA GTG GAA ATG TTA CCA TCC AAA GCT GGA ATT     3360 
Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile 
1105                1110                1115                1120 

TGG CGG GTG GAA TGC CTT ATT GGC GAG CAT CTA CAT GCT GGG ATG AGC     3408 
Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser 
                1125                1130                1135 

ACA CTT TTT CTG GTG TAC TCC AAT AAG TGT CAG ACT CCC CTG GGA ATG     3456 
Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met 
            1140                1145                1150 

GCT TCT GGA CAC ATT AGA GAT TTT CAG ATT ACA GCT TCA GGA CAA TAT     3504 
Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 
        1155                1160                1165 

GGA CAG TGG GCC CCA AAG CTG GCC AGA CTT CAT TAT TCC GGA TCA ATC     3552 
Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile 
    1170                1175                1180 

AAT GCC TGG AGC ACC AAG GAG CCC TTT TCT TGG ATC AAA GTT GAC CTG     3600 
Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu 
1185                1190                1195                1200 

TTG GCA CCA ATG ATT ATT CAC GGC ATC AAG ACC CAG GGT GCC CGT CAG     3648 
Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln 
                1205                1210                1215 

AAG TTC TCC AGC CTC TAC ATC TCT CAA TTT ATC ATC ATG TAT AGT CTC     3696 
Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu 
            1220                1225                1230 

GAT GGG AAG AAG TGG CAG ACT TAT CGA GGA AAT TCC ACT GGA ACC CTC     3744 
Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 
        1235                1240                1245 

ATG GTC TTC TTT GGC AAT GTG GAT TCA TCT GGG ATA AAA CAC AAT ATT     3792 
Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile 
    1250                1255                1260 

TTC AAC CCT CCA ATT ATT GCT CGA TAC ATC CGT TTG CAC CCA ACT CAT     3840 
Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His 
1265                1270                1275                1280 

TAT AGC ATT CGC AGC ACT CTT CGC ATG GAG TTG ATG GGC TGT GAT TTA     3888 
Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu 
                1285                1290                1295 

AAT AGT TGC AGC ATG CCA TTG GGA ATG GAG AGT AAA GCA ATA TCA GAT     3936 
Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp 
            1300                1305                1310 

GCA CAG ATT ACT GCT TCA TCC TAC TTT ACC AAT ATG TTT GCC ACC TGG     3984 
Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp 
        1315                1320                1325 

TCT CCT TCA AAA GCT CGA CTA CAC CTA CAA GGG AGG AGT AAT GCC TGG     4032 
Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp 
    1330                1335                1340 

AGA CCT CAA GTT AAC AAT CCA AAA GAG TGG CTG CAA GTG GAC TTC CAG     4080 
Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln 
1345                1350                1355                1360 

AAG ACA ATG AAA GTC ACA GGA GTA ACT ACT CAG GGA GTA AAA TCT CTG     4128 
Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu 
                1365                1370                1375 

CTT ACC TCT ATG TAC GTG AAG GAG TTC CTC ATA TCG TCG TCG CAA GAT     4176 
Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp 
            1380                1385                1390 

GGC CAT CAG TGG ACT CTC TTT TTT CAA AAT GGC AAA GTA AAA GTT TTC     4224 
Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 
        1395                1400                1405 

CAG GGA AAT CAA GAC TCC TTC ACA CCT GTC GTG AAC TCT CTA GAC CCA     4272 
Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro 
    1410                1415                1420 

CCG TTA CTC ACT CGC TAC CTT CGA ATT CAC CCC CAG AGT TGG GTG CAC     4320 
Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His 
1425                1430                1435                1440 

CAG ATT GCC CTG AGG ATG GAG GTT CTG GGC TGC GAG GCA CAG GAC CTC     4368 
Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu 
                1445                1450                1455 

TAC TGA                                                             4374 
Tyr 

 
           
           
             
               9164 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
             
               CDS  
                1006..5376 

 
             
              2 

GTCGACGGTA TCGATAAGCT TGATATCGAA TTCCTGCAGC CCGGGGGATC CACTAGTACT     60 

CGAGACCTAG GAGTTAATTT TTAAAAAGCA GTCAAAAGTC CAAGTGGCCC TTGCGAGCA     120 

TTACTCTCTC TGTTTGCTCT GGTTAATAAT CTCAGGAGCA CAAACATTCC TTACTAGTC     180 

TAGAAGTTAA TTTTTAAAAA GCAGTCAAAA GTCCAAGTGG CCCTTGCGAG CATTTACTC     240 

CTCTGTTTGC TCTGGTTAAT AATCTCAGGA GCACAAACAT TCCTTACTAG TTCTAGAGC     300 

GCCGCCAGTG TGCTGGAATT CGGCTTTTTT AGGGCTGGAA GCTACCTTTG ACATCATTT     360 

CTCTGCGAAT GCATGTATAA TTTCTACAGA ACCTATTAGA AAGGATCACC CAGCCTCTG     420 

TTTTGTACAA CTTTCCCTTA AAAAACTGCC AATTCCACTG CTGTTTGGCC CAATAGTGA     480 

AACTTTTTCC TGCTGCCTCT TGGTGCTTTT GCCTATGGCC CCTATTCTGC CTGCTGAAG     540 

CACTCTTGCC AGCATGGACT TAAACCCCTC CAGCTCTGAC AATCCTCTTT CTCTTTTGT     600 

TTACATGAAG GGTCTGGCAG CCAAAGCAAT CACTCAAAGT TCAAACCTTA TCATTTTTT     660 

CTTTGTTCCT CTTGGCCTTG GTTTTGTACA TCAGCTTTGA AAATACCATC CCAGGGTTA     720 

TGCTGGGGTT AATTTATAAC TAAGAGTGCT CTAGTTTTGC AATACAGGAC ATGCTATAA     780 

AATGGAAAGA TGTTGCTTTC TGAGAGATCT CGAGGAAGCT AACAACAAAG AACAACAAA     840 

AACAATCAGG TAAGTATCCT TTTTACAGCA CAACTTAATG AGACAGATAG AAACTGGTC     900 

TGTAGAAACA GAGTAGTCGC CTGCTTTTCT GCCAGGTGCT GACTTCTCTC CCCTTCTCT     960 

TTTTCCTTTT CTCAGGATAA CAAGAACGAA ACAATAACAG CCACC ATG GAA ATA       1014 
                                                  Met Glu Ile 
                                                    1 

GAG CTC TCC ACC TGC TTC TTT CTG TGC CTT TTG CGA TTC TGC TTT AGT     1062 
Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe Cys Phe Ser 
      5                  10                  15 

GCC ACC AGA AGA TAC TAC CTG GGT GCA GTG GAA CTG TCA TGG GAC TAT     1110 
Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 
 20                  25                  30                  35 

ATG CAA AGT GAT CTC GGT GAG CTG CCT GTG GAC GCA AGA TTT CCT CCT     1158 
Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 
                 40                  45                  50 

AGA GTG CCA AAA TCT TTT CCA TTC AAC ACC TCA GTC GTG TAC AAA AAG     1206 
Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 
             55                  60                  65 

ACT CTG TTT GTA GAA TTC ACG GTT CAC CTT TTC AAC ATC GCT AAG CCA     1254 
Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro 
         70                  75                  80 

AGG CCA CCC TGG ATG GGT CTG CTA GGT CCT ACC ATC CAG GCT GAG GTT     1302 
Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val 
     85                  90                  95 

TAT GAT ACA GTG GTC ATT ACA CTT AAG AAC ATG GCT TCC CAT CCT GTC     1350 
Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 
100                 105                 110                 115 

AGT CTT CAT GCT GTT GGT GTA TCC TAC TGG AAA GCT TCT GAG GGA GCT     1398 
Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 
                120                 125                 130 

GAA TAT GAT GAT CAG ACC AGT CAA AGG GAG AAA GAA GAT GAT AAA GTC     1446 
Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 
            135                 140                 145 

TTC CCT GGT GGA AGC CAT ACA TAT GTC TGG CAG GTC CTG AAA GAG AAT     1494 
Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 
        150                 155                 160 

GGT CCA ATG GCC TCT GAC CCA CTG TGC CTT ACC TAC TCA TAT CTT TCT     1542 
Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 
    165                 170                 175 

CAT GTG GAC CTG GTA AAA GAC TTG AAT TCA GGC CTC ATT GGA GCC CTA     1590 
His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 
180                 185                 190                 195 

CTA GTA TGT AGA GAA GGG AGT CTG GCC AAG GAA AAG ACA CAG ACC TTG     1638 
Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 
                200                 205                 210 

CAC AAA TTT ATA CTA CTT TTT GCT GTA TTT GAT GAA GGG AAA AGT TGG     1686 
His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 
            215                 220                 225 

CAC TCA GAA ACA AAG AAC TCC TTG ATG CAG GAT AGG GAT GCT GCA TCT     1734 
His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 
        230                 235                 240 

GCT CGG GCC TGG CCT AAA ATG CAC ACA GTC AAT GGT TAT GTA AAC AGG     1782 
Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg 
    245                 250                 255 

TCT CTG CCA GGT CTG ATT GGA TGC CAC AGG AAA TCA GTC TAT TGG CAT     1830 
Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 
260                 265                 270                 275 

GTG ATT GGA ATG GGC ACC ACT CCT GAA GTG CAC TCA ATA TTC CTC GAA     1878 
Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 
                280                 285                 290 

GGT CAC ACA TTT CTT GTG AGG AAC CAT CGC CAG GCG TCC TTG GAA ATC     1926 
Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 
            295                 300                 305 

TCG CCA ATA ACT TTC CTT ACT GCT CAA ACA CTC TTG ATG GAC CTT GGA     1974 
Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 
        310                 315                 320 

CAG TTT CTA CTG TTT TGT CAT ATC TCT TCC CAC CAA CAT GAT GGC ATG     2022 
Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met 
    325                 330                 335 

GAA GCT TAT GTC AAA GTA GAC AGC TGT CCA GAG GAA CCC CAA CTA CGA     2070 
Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 
340                 345                 350                 355 

ATG AAA AAT AAT GAA GAA GCG GAA GAC TAT GAT GAT GAT CTT ACT GAT     2118 
Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 
                360                 365                 370 

TCT GAA ATG GAT GTG GTC AGG TTT GAT GAT GAC AAC TCT CCT TCC TTT     2166 
Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 
            375                 380                 385 

ATC CAA ATT CGC TCA GTT GCC AAG AAG CAT CCT AAA ACT TGG GTA CAT     2214 
Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 
        390                 395                 400 

TAC ATT GCT GCT GAA GAG GAG GAC TGG GAC TAT GCT CCC TTA GTC CTC     2262 
Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 
    405                 410                 415 

GCC CCC GAT GAC AGA AGT TAT AAA AGT CAA TAT TTG AAC AAT GGC CCT     2310 
Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 
420                 425                 430                 435 

CAG CGG ATT GGT AGG AAG TAC AAA AAA GTC CGA TTT ATG GCA TAC ACA     2358 
Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 
                440                 445                 450 

GAT GAA ACC TTT AAG ACT CGT GAA GCT ATT CAG CAT GAA TCA GGA ATC     2406 
Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 
            455                 460                 465 

TTG GGA CCT TTA CTT TAT GGG GAA GTT GGA GAC ACA CTG TTG ATT ATA     2454 
Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 
        470                 475                 480 

TTT AAG AAT CAA GCA AGC AGA CCA TAT AAC ATC TAC CCT CAC GGA ATC     2502 
Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 
    485                 490                 495 

ACT GAT GTC CGT CCT TTG TAT TCA AGG AGA TTA CCA AAA GGT GTA AAA     2550 
Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 
500                 505                 510                 515 

CAT TTG AAG GAT TTT CCA ATT CTG CCA GGA GAA ATA TTC AAA TAT AAA     2598 
His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 
                520                 525                 530 

TGG ACA GTG ACT GTA GAA GAT GGG CCA ACT AAA TCA GAT CCT CGG TGC     2646 
Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 
            535                 540                 545 

CTG ACC CGC TAT TAC TCT AGT TTC GTT AAT ATG GAG AGA GAT CTA GCT     2694 
Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 
        550                 555                 560 

TCA GGA CTC ATT GGC CCT CTC CTC ATC TGC TAC AAA GAA TCT GTA GAT     2742 
Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp 
    565                 570                 575 

CAA AGA GGA AAC CAG ATA ATG TCA GAC AAG AGG AAT GTC ATC CTG TTT     2790 
Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 
580                 585                 590                 595 

TCT GTA TTT GAT GAG AAC CGA AGC TGG TAC CTC ACA GAG AAT ATA CAA     2838 
Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 
                600                 605                 610 

CGC TTT CTC CCC AAT CCA GCT GGA GTG CAG CTT GAG GAT CCA GAG TTC     2886 
Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 
            615                 620                 625 

CAA GCC TCC AAC ATC ATG CAC AGC ATC AAT GGC TAT GTT TTT GAT AGT     2934 
Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 
        630                 635                 640 

TTG CAG TTG TCA GTT TGT TTG CAT GAG GTG GCA TAC TGG TAC ATT CTA     2982 
Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 
    645                 650                 655 

AGC ATT GGA GCA CAG ACT GAC TTC CTT TCT GTC TTC TTC TCT GGA TAT     3030 
Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 
660                 665                 670                 675 

ACC TTC AAA CAC AAA ATG GTC TAT GAA GAC ACA CTC ACC CTA TTC CCA     3078 
Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 
                680                 685                 690 

TTC TCA GGA GAA ACT GTC TTC ATG TCG ATG GAA AAC CCA GGT CTA TGG     3126 
Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 
            695                 700                 705 

ATT CTG GGG TGC CAC AAC TCA GAC TTT CGG AAC AGA GGC ATG ACC GCC     3174 
Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 
        710                 715                 720 

TTA CTG AAG GTT TCT AGT TGT GAC AAG AAC ACT GGT GAT TAT TAC GAG     3222 
Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 
    725                 730                 735 

GAC AGT TAT GAA GAT ATT TCA GCA TAC TTG CTG AGT AAA AAC AAT GCC     3270 
Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 
740                 745                 750                 755 

ATT GAA CCA AGA AGC TTC TCC CAG AAC CCA CCA GTC TTG AAA CGC CAT     3318 
Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His 
                760                 765                 770 

CAA CGG GAA ATA ACT CGT ACT ACT CTT CAG TCA GAT CAA GAG GAA ATT     3366 
Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile 
            775                 780                 785 

GAC TAT GAT GAT ACC ATA TCA GTT GAA ATG AAG AAG GAA GAT TTT GAC     3414 
Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp 
        790                 795                 800 

ATT TAT GAT GAG GAT GAA AAT CAG AGC CCC CGC AGC TTT CAA AAG AAA     3462 
Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys 
    805                 810                 815 

ACA CGA CAC TAT TTT ATT GCT GCA GTG GAG AGG CTC TGG GAT TAT GGG     3510 
Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly 
820                 825                 830                 835 

ATG AGT AGC TCC CCA CAT GTT CTA AGA AAC AGG GCT CAG AGT GGC AGT     3558 
Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser 
                840                 845                 850 

GTC CCT CAG TTC AAG AAA GTT GTT TTC CAG GAA TTT ACT GAT GGC TCC     3606 
Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser 
            855                 860                 865 

TTT ACT CAG CCC TTA TAC CGT GGA GAA CTA AAT GAA CAT TTG GGA CTC     3654 
Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 
        870                 875                 880 

CTG GGG CCA TAT ATA AGA GCA GAA GTT GAA GAT AAT ATC ATG GTA ACT     3702 
Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr 
    885                 890                 895 

TTC AGA AAT CAG GCC TCT CGT CCC TAT TCC TTC TAT TCT AGC CTT ATT     3750 
Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile 
900                 905                 910                 915 

TCT TAT GAG GAA GAT CAG AGG CAA GGA GCA GAA CCT AGA AAA AAC TTT     3798 
Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe 
                920                 925                 930 

GTC AAG CCT AAT GAA ACC AAA ACT TAC TTT TGG AAA GTG CAA CAT CAT     3846 
Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His 
            935                 940                 945 

ATG GCA CCC ACT AAA GAT GAG TTT GAC TGC AAA GCC TGG GCT TAT TTC     3894 
Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe 
        950                 955                 960 

TCT GAT GTT GAC CTG GAA AAA GAT GTG CAC TCA GGC CTG ATT GGA CCC     3942 
Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro 
    965                 970                 975 

CTT CTG GTC TGC CAC ACT AAC ACA CTG AAC CCT GCT CAT GGG AGA CAA     3990 
Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln 
980                 985                 990                 995 

GTG ACA GTA CAG GAA TTT GCT CTG TTT TTC ACC ATC TTT GAT GAG ACC     4038 
Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr 
                1000                1005                1010 

AAA AGC TGG TAC TTC ACT GAA AAT ATG GAA AGA AAC TGC AGG GCT CCC     4086 
Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro 
            1015                1020                1025 

TGC AAT ATC CAG ATG GAA GAT CCC ACT TTT AAA GAG AAT TAT CGC TTC     4134 
Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe 
        1030                1035                1040 

CAT GCA ATC AAT GGC TAC ATA ATG GAT ACA CTA CCT GGC TTA GTA ATG     4182 
His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met 
    1045                1050                1055 

GCT CAG GAT CAA AGG ATT CGA TGG TAT CTG CTC AGC ATG GGC AGC AAT     4230 
Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn 
1060                1065                1070                1075 

GAA AAC ATC CAT TCT ATT CAT TTC AGT GGA CAT GTG TTC ACT GTA CGA     4278 
Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg 
                1080                1085                1090 

AAA AAA GAG GAG TAT AAA ATG GCA CTG TAC AAT CTC TAT CCA GGT GTT     4326 
Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val 
            1095                1100                1105 

TTT GAG ACA GTG GAA ATG TTA CCA TCC AAA GCT GGA ATT TGG CGG GTG     4374 
Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val 
        1110                1115                1120 

GAA TGC CTT ATT GGC GAG CAT CTA CAT GCT GGG ATG AGC ACA CTT TTT     4422 
Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe 
    1125                1130                1135 

CTG GTG TAC AGC AAT AAG TGT CAG ACT CCC CTG GGA ATG GCT TCT GGA     4470 
Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly 
1140                1145                1150                1155 

CAC ATT AGA GAT TTT CAG ATT ACA GCT TCA GGA CAA TAT GGA CAG TGG     4518 
His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp 
                1160                1165                1170 

GCC CCA AAG CTG GCC AGA CTT CAT TAT TCC GGA TCA ATC AAT GCC TGG     4566 
Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp 
            1175                1180                1185 

AGC ACC AAG GAG CCC TTT TCT TGG ATC AAG GTG GAT CTG TTG GCA CCA     4614 
Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 
        1190                1195                1200 

ATG ATT ATT CAC GGC ATC AAG ACC CAG GGT GCC CGT CAG AAG TTC TCC     4662 
Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser 
    1205                1210                1215 

AGC CTC TAC ATC TCT CAG TTT ATC ATC ATG TAT AGT CTT GAT GGG AAG     4710 
Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys 
1220                1225                1230                1235 

AAG TGG CAG ACT TAT CGA GGA AAT TCC ACT GGA ACC TTA ATG GTC TTC     4758 
Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe 
                1240                1245                1250 

TTT GGC AAT GTG GAT TCA TCT GGG ATA AAA CAC AAT ATT TTT AAC CCT     4806 
Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro 
            1255                1260                1265 

CCA ATT ATT GCT CGA TAC ATC CGT TTG CAC CCA ACT CAT TAT AGC ATT     4854 
Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile 
        1270                1275                1280 

CGC AGC ACT CTT CGC ATG GAG TTG ATG GGC TGT GAT TTA AAT AGT TGC     4902 
Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys 
    1285                1290                1295 

AGC ATG CCA TTG GGA ATG GAG AGT AAA GCA ATA TCA GAT GCA CAG ATT     4950 
Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile 
1300                1305                1310                1315 

ACT GCT TCA TCC TAC TTT ACC AAT ATG TTT GCC ACC TGG TCT CCT TCA     4998 
Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser 
                1320                1325                1330 

AAA GCT CGA CTT CAC CTC CAA GGG AGG AGT AAT GCC TGG AGA CCT CAG     5046 
Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln 
            1335                1340                1345 

GTG AAT AAT CCA AAA GAG TGG CTG CAA GTG GAC TTC CAG AAG ACA ATG     5094 
Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met 
        1350                1355                1360 

AAA GTC ACA GGA GTA ACT ACT CAG GGA GTA AAA TCT CTG CTT ACC AGC     5142 
Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser 
    1365                1370                1375 

ATG TAT GTG AAG GAG TTC CTC ATC TCC AGC AGT CAA GAT GGC CAT CAG     5190 
Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln 
1380                1385                1390                1395 

TGG ACT CTC TTT TTT CAG AAT GGC AAA GTA AAG GTT TTT CAG GGA AAT     5238 
Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn 
                1400                1405                1410 

CAA GAC TCC TTC ACA CCT GTG GTG AAC TCT CTA GAC CCA CCG TTA CTG     5286 
Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu 
            1415                1420                1425 

ACT CGC TAC CTT CGA ATT CAC CCC CAG AGT TGG GTG CAC CAG ATT GCC     5334 
Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 
        1430                1435                1440 

CTG AGG ATG GAG GTT CTG GGC TGC GAG GCA CAG GAC CTC TAC             5376 
Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 
    1445                1450                1455 

TGAGGGTGGC CACTGCAGCA CCTGCCACTG CCGTCACCTC TCCCTCCTCA GCTCCAGG     5436 

AGTGTCCCTC CCTGGCTTGC CTTCTACCTT TGTGCTAAAT CCTAGCAGAC ACTGCCTT     5496 

AGCCTCCTGA ATTAACTATC ATCAGTCCTG CATTTCTTTG GTGGGGGGCC AGGAGGGT     5556 

ATCCAATTTA ACTTAACTCT TACCTATTTT CTGCAGCTGC TCCCAGATTA CTCCTTCC     5616 

CCAATATAAC TAGGCAAAAA GAAGTGAGGA GAAACCTGCA TGAAAGCATT CTTCCCTG     5676 

AAGTTAGGCC TCTCAGAGTC ACCACTTCCT CTGTTGTAGA AAAACTATGT GATGAAAC     5736 

TGAAAAAGAT ATTTATGATG TTAACTTGTT TATTGCAGCT TATAATGGTT ACAAATAA     5796 

CAATAGCATC ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGT     5856 

GTCCAAACTC ATCAATGTAT CTTATCATGT CTGGATCCCC GGGTGGCATC CCTGTGAC     5916 

CTCCCCAGTG CCTCTCCTGG CCCTGGAAGT TGCCACTCCA GTGCCCACCA GCCTTGTC     5976 

AATAAAATTA AGTTGCATCA TTTTGTCTGA CTAGGTGTCC TTCTATAATA TTATGGGG     6036 

GAGGGGGGTG GTATGGAGCA AGGGGCAAGT TGGGAAGACA ACCTGTAGGG CCTGCGGG     6096 

CTATTCGGGA ACCAAGCTGG AGTGCAGTGG CACAATCTTG GCTCACTGCA ATCTCCGC     6156 

CCTGGGTTCA AGCGATTCTC CTGCCTCAGC CTCCCGAGTT GTTGGGATTC CAGGCATG     6216 

TGACCAGGCT CAGCTAATTT TTGTTTTTTT GGTAGAGACG GGGTTTCACC ATATTGGC     6276 

GGCTGGTCTC CAACTCCTAA TCTCAGGTGA TCTACCCACC TTGGCCTCCC AAATTGCT     6336 

GATTACAGGC GTGAACCACT GCTCCCTTCC CTGTCCTTCT GATTTTAAAA TAACTATA     6396 

AGCAGGAGGA CGTCCAGACA CAGCATAGGC TACCTGCCAT GCCCAACCGG TGGGACAT     6456 

GAGTTGCTTG CTTGGCACTG TCCTCTCATG CGTTGGGTCC ACTCAGTAGA TGCCTGTT     6516 

ATTCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAATTG TTATCCGCTC ACAATTCC     6576 

ACAACATACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA GTGAGCTA     6636 

TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG TCGTGCCA     6696 

TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCTCTTC     6756 

CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGC     6816 

ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACAT     6876 

GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTT     6936 

ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCG     6996 

ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTC     7056 

CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGT     7116 

CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAA     7176 

TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTA     7236 

GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAA     7296 

GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAA     7356 

ACGGCTACAC TAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTT     7416 

GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTT     7476 

TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGAT     7536 

TTTCTACGGG GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCAT     7596 

GATTATCAAA AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATC     7656 

TCTAAAGTAT ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGC     7716 

CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTA     7776 

TAACTACGAT ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGA     7836 

CACGCTCACC GGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCG     7896 

GAAGTGGTCC TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGC     7956 

GAGTAAGTAG TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCAT     8016 

TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAG     8076 

GAGTTACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGAT     8136 

TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAA     8196 

CTCTTACTGT CATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAA     8256 

CATTCTGAGA ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGA     8316 

ATACCGCGCC ACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGG     8376 

GAAAACTCTC AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGC     8436 

CCAACTGATC TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGG     8496 

GGCAAAATGC CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACT     8556 

TCCTTTTTCA ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACAT     8616 

TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGT     8676 

CACCTGACGT CTAAGAAACC ATTATTATCA TGACATTAAC CTATAAAAAT AGGCGTAT     8736 

CGAGGCCCTT TCGTCTCGCG CGTTTCGGTG ATGACGGTGA AAACCTCTGA CACATGCA     8796 

TCCCGGAGAC GGTCACAGCT TGTCTGTAAG CGGATGCCGG GAGCAGACAA GCCCGTCA     8856 

GCGCGTCAGC GGGTGTTGGC GGGTGTCGGG GCTGGCTTAA CTATGCGGCA TCAGAGCA     8916 

TTGTACTGAG AGTGCACCAT ATGCGGTGTG AAATACCGCA CAGATGCGTA AGGAGAAA     8976 

ACCGCATCAG GCGCCATTCG CCATTCAGGC TGCGCAACTG TTGGGAAGGG CGATCGGT     9036 

GGGCCTCTTC GCTATTACGC CAGCTGGCGA AAGGGGGATG TGCTGCAAGG CGATTAAG     9096 

GGGTAACGCC AGGGTTTTCC CAGTCACGAC GTTGTAAAAC GACGGCCAGT GCCAAGCT     9156 

GGCTGCAG                                                            9164 

 
           
           
             
               12022 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
             
               CDS  
                1006..3294 

 
             
             
               CDS  
                6153..8234 

 
             
              3 

GTCGACGGTA TCGATAAGCT TGATATCGAA TTCCTGCAGC CCGGGGGATC CACTAGTACT     60 

CGAGACCTAG GAGTTAATTT TTAAAAAGCA GTCAAAAGTC CAAGTGGCCC TTGCGAGCA     120 

TTACTCTCTC TGTTTGCTCT GGTTAATAAT CTCAGGAGCA CAAACATTCC TTACTAGTC     180 

TAGAAGTTAA TTTTTAAAAA GCAGTCAAAA GTCCAAGTGG CCCTTGCGAG CATTTACTC     240 

CTCTGTTTGC TCTGGTTAAT AATCTCAGGA GCACAAACAT TCCTTACTAG TTCTAGAGC     300 

GCCGCCAGTG TGCTGGAATT CGGCTTTTTT AGGGCTGGAA GCTACCTTTG ACATCATTT     360 

CTCTGCGAAT GCATGTATAA TTTCTACAGA ACCTATTAGA AAGGATCACC CAGCCTCTG     420 

TTTTGTACAA CTTTCCCTTA AAAAACTGCC AATTCCACTG CTGTTTGGCC CAATAGTGA     480 

AACTTTTTCC TGCTGCCTCT TGGTGCTTTT GCCTATGGCC CCTATTCTGC CTGCTGAAG     540 

CACTCTTGCC AGCATGGACT TAAACCCCTC CAGCTCTGAC AATCCTCTTT CTCTTTTGT     600 

TTACATGAAG GGTCTGGCAG CCAAAGCAAT CACTCAAAGT TCAAACCTTA TCATTTTTT     660 

CTTTGTTCCT CTTGGCCTTG GTTTTGTACA TCAGCTTTGA AAATACCATC CCAGGGTTA     720 

TGCTGGGGTT AATTTATAAC TAAGAGTGCT CTAGTTTTGC AATACAGGAC ATGCTATAA     780 

AATGGAAAGA TGTTGCTTTC TGAGAGATCT CGAGGAAGCT AACAACAAAG AACAACAAA     840 

AACAATCAGG TAAGTATCCT TTTTACAGCA CAACTTAATG AGACAGATAG AAACTGGTC     900 

TGTAGAAACA GAGTAGTCGC CTGCTTTTCT GCCAGGTGCT GACTTCTCTC CCCTTCTCT     960 

TTTTCCTTTT CTCAGGATAA CAAGAACGAA ACAATAACAG CCACC ATG GAA ATA       1014 
                                                  Met Glu Ile 
                                                    1 

GAG CTC TCC ACC TGC TTC TTT CTG TGC CTT TTG CGA TTC TGC TTT AGT     1062 
Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe Cys Phe Ser 
      5                  10                  15 

GCC ACC AGA AGA TAC TAC CTG GGT GCA GTG GAA CTG TCA TGG GAC TAT     1110 
Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 
 20                  25                  30                  35 

ATG CAA AGT GAT CTC GGT GAG CTG CCT GTG GAC GCA AGA TTT CCT CCT     1158 
Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 
                 40                  45                  50 

AGA GTG CCA AAA TCT TTT CCA TTC AAC ACC TCA GTC GTG TAC AAA AAG     1206 
Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 
             55                  60                  65 

ACT CTG TTT GTA GAA TTC ACG GTT CAC CTT TTC AAC ATC GCT AAG CCA     1254 
Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro 
         70                  75                  80 

AGG CCA CCC TGG ATG GGT CTG CTA GGT CCT ACC ATC CAG GCT GAG GTT     1302 
Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val 
     85                  90                  95 

TAT GAT ACA GTG GTC ATT ACA CTT AAG AAC ATG GCT TCC CAT CCT GTC     1350 
Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 
100                 105                 110                 115 

AGT CTT CAT GCT GTT GGT GTA TCC TAC TGG AAA GCT TCT GAG GGA GCT     1398 
Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 
                120                 125                 130 

GAA TAT GAT GAT CAG ACC AGT CAA AGG GAG AAA GAA GAT GAT AAA GTC     1446 
Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 
            135                 140                 145 

TTC CCT GGT GGA AGC CAT ACA TAT GTC TGG CAG GTC CTG AAA GAG AAT     1494 
Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 
        150                 155                 160 

GGT CCA ATG GCC TCT GAC CCA CTG TGC CTT ACC TAC TCA TAT CTT TCT     1542 
Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 
    165                 170                 175 

CAT GTG GAC CTG GTA AAA GAC TTG AAT TCA GGC CTC ATT GGA GCC CTA     1590 
His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 
180                 185                 190                 195 

CTA GTA TGT AGA GAA GGG AGT CTG GCC AAG GAA AAG ACA CAG ACC TTG     1638 
Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 
                200                 205                 210 

CAC AAA TTT ATA CTA CTT TTT GCT GTA TTT GAT GAA GGG AAA AGT TGG     1686 
His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 
            215                 220                 225 

CAC TCA GAA ACA AAG AAC TCC TTG ATG CAG GAT AGG GAT GCT GCA TCT     1734 
His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 
        230                 235                 240 

GCT CGG GCC TGG CCT AAA ATG CAC ACA GTC AAT GGT TAT GTA AAC AGG     1782 
Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg 
    245                 250                 255 

TCT CTG CCA GGT CTG ATT GGA TGC CAC AGG AAA TCA GTC TAT TGG CAT     1830 
Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 
260                 265                 270                 275 

GTG ATT GGA ATG GGC ACC ACT CCT GAA GTG CAC TCA ATA TTC CTC GAA     1878 
Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 
                280                 285                 290 

GGT CAC ACA TTT CTT GTG AGG AAC CAT CGC CAG GCG TCC TTG GAA ATC     1926 
Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 
            295                 300                 305 

TCG CCA ATA ACT TTC CTT ACT GCT CAA ACA CTC TTG ATG GAC CTT GGA     1974 
Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 
        310                 315                 320 

CAG TTT CTA CTG TTT TGT CAT ATC TCT TCC CAC CAA CAT GAT GGC ATG     2022 
Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met 
    325                 330                 335 

GAA GCT TAT GTC AAA GTA GAC AGC TGT CCA GAG GAA CCC CAA CTA CGA     2070 
Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 
340                 345                 350                 355 

ATG AAA AAT AAT GAA GAA GCG GAA GAC TAT GAT GAT GAT CTT ACT GAT     2118 
Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 
                360                 365                 370 

TCT GAA ATG GAT GTG GTC AGG TTT GAT GAT GAC AAC TCT CCT TCC TTT     2166 
Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 
            375                 380                 385 

ATC CAA ATT CGC TCA GTT GCC AAG AAG CAT CCT AAA ACT TGG GTA CAT     2214 
Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 
        390                 395                 400 

TAC ATT GCT GCT GAA GAG GAG GAC TGG GAC TAT GCT CCC TTA GTC CTC     2262 
Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 
    405                 410                 415 

GCC CCC GAT GAC AGA AGT TAT AAA AGT CAA TAT TTG AAC AAT GGC CCT     2310 
Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 
420                 425                 430                 435 

CAG CGG ATT GGT AGG AAG TAC AAA AAA GTC CGA TTT ATG GCA TAC ACA     2358 
Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 
                440                 445                 450 

GAT GAA ACC TTT AAG ACT CGT GAA GCT ATT CAG CAT GAA TCA GGA ATC     2406 
Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 
            455                 460                 465 

TTG GGA CCT TTA CTT TAT GGG GAA GTT GGA GAC ACA CTG TTG ATT ATA     2454 
Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 
        470                 475                 480 

TTT AAG AAT CAA GCA AGC AGA CCA TAT AAC ATC TAC CCT CAC GGA ATC     2502 
Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 
    485                 490                 495 

ACT GAT GTC CGT CCT TTG TAT TCA AGG AGA TTA CCA AAA GGT GTA AAA     2550 
Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 
500                 505                 510                 515 

CAT TTG AAG GAT TTT CCA ATT CTG CCA GGA GAA ATA TTC AAA TAT AAA     2598 
His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 
                520                 525                 530 

TGG ACA GTG ACT GTA GAA GAT GGG CCA ACT AAA TCA GAT CCT CGG TGC     2646 
Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 
            535                 540                 545 

CTG ACC CGC TAT TAC TCT AGT TTC GTT AAT ATG GAG AGA GAT CTA GCT     2694 
Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 
        550                 555                 560 

TCA GGA CTC ATT GGC CCT CTC CTC ATC TGC TAC AAA GAA TCT GTA GAT     2742 
Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp 
    565                 570                 575 

CAA AGA GGA AAC CAG ATA ATG TCA GAC AAG AGG AAT GTC ATC CTG TTT     2790 
Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 
580                 585                 590                 595 

TCT GTA TTT GAT GAG AAC CGA AGC TGG TAC CTC ACA GAG AAT ATA CAA     2838 
Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 
                600                 605                 610 

CGC TTT CTC CCC AAT CCA GCT GGA GTG CAG CTT GAG GAT CCA GAG TTC     2886 
Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 
            615                 620                 625 

CAA GCC TCC AAC ATC ATG CAC AGC ATC AAT GGC TAT GTT TTT GAT AGT     2934 
Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 
        630                 635                 640 

TTG CAG TTG TCA GTT TGT TTG CAT GAG GTG GCA TAC TGG TAC ATT CTA     2982 
Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 
    645                 650                 655 

AGC ATT GGA GCA CAG ACT GAC TTC CTT TCT GTC TTC TTC TCT GGA TAT     3030 
Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 
660                 665                 670                 675 

ACC TTC AAA CAC AAA ATG GTC TAT GAA GAC ACA CTC ACC CTA TTC CCA     3078 
Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 
                680                 685                 690 

TTC TCA GGA GAA ACT GTC TTC ATG TCG ATG GAA AAC CCA GGT CTA TGG     3126 
Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 
            695                 700                 705 

ATT CTG GGG TGC CAC AAC TCA GAC TTT CGG AAC AGA GGC ATG ACC GCC     3174 
Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 
        710                 715                 720 

TTA CTG AAG GTT TCT AGT TGT GAC AAG AAC ACT GGT GAT TAT TAC GAG     3222 
Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 
    725                 730                 735 

GAC AGT TAT GAA GAT ATT TCA GCA TAC TTG CTG AGT AAA AAC AAT GCC     3270 
Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 
740                 745                 750                 755 

ATT GAA CCA AGA AGC TTC TCC CAG GTAAGTTATT ATATAAATTC AAGACACCC     3324 
Ile Glu Pro Arg Ser Phe Ser Gln 
                760 

AGCACTAGGC AAAAGCAATT TAATGCCACC ACAATTCCAG AAAATGACAT AGAGAAGA     3384 

GACCCTTGGT TTGCACACAG AACACCTATG CCTAAAATAC AAAATGTCTC CTCTAGTG     3444 

TTGTTGATGC TCTTGCGACA GAGTCCTACT CCACATGGGC TATCCTTATC TGATCTCC     3504 

GAAGCCAAAT ATGAGACTTT TTCTGATGAT CCATCACCTG GAGCAATAGA CAGTAATA     3564 

AGCCTGTCTG AAATGACACA CTTCAGGCCA CAGCTCCATC ACAGTGGGGA CATGGTAT     3624 

ACCCCTGAGT CAGGCCTCCA ATTAAGATTA AATGAGAAAC TGGGGACAAC TGCAGCAA     3684 

GAGTTGAAGA AACTTGATTT CAAAGTTTCT AGTACATCAA ATAATCTGAT TTCAACAA     3744 

CCATCAGACA ATTTGGCAGC AGGTACTGAT AATACAAGTT CCTTAGGACC CCCAAGTA     3804 

CCAGTTCATT ATGATAGTCA ATTAGATACC ACTCTATTTG GCAAAAAGTC ATCTCCCC     3864 

ACTGAGTCTG GTGGACCTCT GAGCTTGAGT GAAGAAAATA ATGATTCAAA GTTGTTAG     3924 

TCAGGTTTAA TGAATAGCCA AGAAAGTTCA TGGGGAAAAA ATGTATCGTC AACAGAGA     3984 

GGTAGGTTAT TTAAAGGGAA AAGAGCTCAT GGACCTGCTT TGTTGACTAA AGATAATG     4044 

TTATTCAAAG TTAGCATCTC TTTGTTAAAG ACAAACAAAA CTTCCAATAA TTCAGCAA     4104 

AATAGAAAGA CTCACATTGA TGGCCCATCA TTATTAATTG AGAATAGTCC ATCAGTCT     4164 

CAAAATATAT TAGAAAGTGA CACTGAGTTT AAAAAAGTGA CACCTTTGAT TCATGACA     4224 

ATGCTTATGG ACAAAAATGC TACAGCTTTG AGGCTAAATC ATATGTCAAA TAAAACTA     4284 

TCATCAAAAA ACATGGAAAT GGTCCAACAG AAAAAAGAGG GCCCCATTCC ACCAGATG     4344 

CAAAATCCAG ATATGTCGTT CTTTAAGATG CTATTCTTGC CAGAATCAGC AAGGTGGA     4404 

CAAAGGACTC ATGGAAAGAA CTCTCTGAAC TCTGGGCAAG GCCCCAGTCC AAAGCAAT     4464 

GTATCCTTAG GACCAGAAAA ATCTGTGGAA GGTCAGAATT TCTTGTCTGA GAAAAACA     4524 

GTGGTAGTAG GAAAGGGTGA ATTTACAAAG GACGTAGGAC TCAAAGAGAT GGTTTTTC     4584 

AGCAGCAGAA ACCTATTTCT TACTAACTTG GATAATTTAC ATGAAAATAA TACACACA     4644 

CAAGAAAAAA AAATTCAGGA AGAAATAGAA AAGAAGGAAA CATTAATCCA AGAGAATG     4704 

GTTTTGCCTC AGATACATAC AGTGACTGGC ACTAAGAATT TCATGAAGAA CCTTTTCT     4764 

CTGAGCACTA GGCAAAATGT AGAAGGTTCA TATGAGGGGG CATATGCTCC AGTACTTC     4824 

GATTTTAGGT CATTAAATGA TTCAACAAAT AGAACAAAGA AACACACAGC TCATTTCT     4884 

AAAAAAGGGG AGGAAGAAAA CTTGGAAGGC TTGGGAAATC AAACCAAGCA AATTGTAG     4944 

AAATATGCAT GCACCACAAG GATATCTCCT AATACAAGCC AGCAGAATTT TGTCACGC     5004 

CGTAGTAAGA GAGCTTTGAA ACAATTCAGA CTCCCACTAG AAGAAACAGA ACTTGAAA     5064 

AGGATAATTG TGGATGACAC CTCAACCCAG TGGTCCAAAA ACATGAAACA TTTGACCC     5124 

AGCACCCTCA CACAGATAGA CTACAATGAG AAGGAGAAAG GGGCCATTAC TCAGTCTC     5184 

TTATCAGATT GCCTTACGAG GAGTCATAGC ATCCCTCAAG CAAATAGATC TCCATTAC     5244 

ATTGCAAAGG TATCATCATT TCCATCTATT AGACCTATAT ATCTGACCAG GGTCCTAT     5304 

CAAGACAACT CTTCTCATCT TCCAGCAGCA TCTTATAGAA AGAAAGATTC TGGGGTCC     5364 

GAAAGCAGTC ATTTCTTACA AGGAGCCAAA AAAAATAACC TTTCTTTAGC CATTCTAA     5424 

TTGGAGATGA CTGGTGATCA AAGAGAGGTT GGCTCCCTGG GGACAAGTGC CACAAATT     5484 

GTCACATACA AGAAAGTTGA GAACACTGTT CTCCCGAAAC CAGACTTGCC CAAAACAT     5544 

GGCAAAGTTG AATTGCTTCC AAAAGTTCAC ATTTATCAGA AGGACCTATT CCCTACGG     5604 

ACTAGCAATG GGTCTCCTGG CCATCTGGAT CTCGTGGAAG GGAGCCTTCT TCAGGGAA     5664 

GAGGGAGCGA TTAAGTGGAA TGAAGCAAAC AGACCTGGAA AAGTTCCCTT TCTGAGAG     5724 

GCAACAGAAA GCTCTGCAAA GACTCCCTCC AAGCTATTGG ATCCTCTTGC TTGGGATA     5784 

CACTATGGTA CTCAGATACC AAAAGAAGAG TGGAAATCCC AAGAGAAGTC ACCAGAAA     5844 

ACAGCTTTTA AGAAAAAGGA TACCATTTTG TCCCTGAACG CTTGTGAAAG CAATCATG     5904 

ATAGCAGCAA TAAATGAGGG ACAAAATAAG CCCGAAATAG AAGTCACCTG GGCAAAGC     5964 

GGTAGGACTG AAAGGCTGTG CTCTCAATTG TGCTAATAAA GCTTGGCAAG AGTATTTC     6024 

GGAAGATGAA GTCATTAACT ATGCAAAATG CTTCTCAGGC ACCTAGGAAA ATGAGGAT     6084 

GAGGCATTTC TACCCACTTG GTACATAAAA TTATTGGGTC ACCCTTTTCC TCTTCTTT     6144 

TTCTCCAG AAC CCA CCA GTC TTG AAA CGC CAT CAA CGG GAA ATA ACT CG     6194 
         Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg 
           1               5                  10 

ACT ACT CTT CAG TCA GAT CAA GAG GAA ATT GAC TAT GAT GAT ACC ATA     6242 
Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 
 15                  20                  25                  30 

TCA GTT GAA ATG AAG AAG GAA GAT TTT GAC ATT TAT GAT GAG GAT GAA     6290 
Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu 
                 35                  40                  45 

AAT CAG AGC CCC CGC AGC TTT CAA AAG AAA ACA CGA CAC TAT TTT ATT     6338 
Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile 
             50                  55                  60 

GCT GCA GTG GAG AGG CTC TGG GAT TAT GGG ATG AGT AGC TCC CCA CAT     6386 
Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His 
         65                  70                  75 

GTT CTA AGA AAC AGG GCT CAG AGT GGC AGT GTC CCT CAG TTC AAG AAA     6434 
Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys 
     80                  85                  90 

GTT GTT TTC CAG GAA TTT ACT GAT GGC TCC TTT ACT CAG CCC TTA TAC     6482 
Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr 
 95                 100                 105                 110 

CGT GGA GAA CTA AAT GAA CAT TTG GGA CTC CTG GGG CCA TAT ATA AGA     6530 
Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg 
                115                 120                 125 

GCA GAA GTT GAA GAT AAT ATC ATG GTA ACT TTC AGA AAT CAG GCC TCT     6578 
Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser 
            130                 135                 140 

CGT CCC TAT TCC TTC TAT TCT AGC CTT ATT TCT TAT GAG GAA GAT CAG     6626 
Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln 
        145                 150                 155 

AGG CAA GGA GCA GAA CCT AGA AAA AAC TTT GTC AAG CCT AAT GAA ACC     6674 
Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr 
    160                 165                 170 

AAA ACT TAC TTT TGG AAA GTG CAA CAT CAT ATG GCA CCC ACT AAA GAT     6722 
Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp 
175                 180                 185                 190 

GAG TTT GAC TGC AAA GCC TGG GCT TAT TTC TCT GAT GTT GAC CTG GAA     6770 
Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu 
                195                 200                 205 

AAA GAT GTG CAC TCA GGC CTG ATT GGA CCC CTT CTG GTC TGC CAC ACT     6818 
Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr 
            210                 215                 220 

AAC ACA CTG AAC CCT GCT CAT GGG AGA CAA GTG ACA GTA CAG GAA TTT     6866 
Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe 
        225                 230                 235 

GCT CTG TTT TTC ACC ATC TTT GAT GAG ACC AAA AGC TGG TAC TTC ACT     6914 
Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr 
    240                 245                 250 

GAA AAT ATG GAA AGA AAC TGC AGG GCT CCC TGC AAT ATC CAG ATG GAA     6962 
Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 
255                 260                 265                 270 

GAT CCC ACT TTT AAA GAG AAT TAT CGC TTC CAT GCA ATC AAT GGC TAC     7010 
Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr 
                275                 280                 285 

ATA ATG GAT ACA CTA CCT GGC TTA GTA ATG GCT CAG GAT CAA AGG ATT     7058 
Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile 
            290                 295                 300 

CGA TGG TAT CTG CTC AGC ATG GGC AGC AAT GAA AAC ATC CAT TCT ATT     7106 
Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile 
        305                 310                 315 

CAT TTC AGT GGA CAT GTG TTC ACT GTA CGA AAA AAA GAG GAG TAT AAA     7154 
His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys 
    320                 325                 330 

ATG GCA CTG TAC AAT CTC TAT CCA GGT GTT TTT GAG ACA GTG GAA ATG     7202 
Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 
335                 340                 345                 350 

TTA CCA TCC AAA GCT GGA ATT TGG CGG GTG GAA TGC CTT ATT GGC GAG     7250 
Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu 
                355                 360                 365 

CAT CTA CAT GCT GGG ATG AGC ACA CTT TTT CTG GTG TAC AGC AAT AAG     7298 
His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys 
            370                 375                 380 

TGT CAG ACT CCC CTG GGA ATG GCT TCT GGA CAC ATT AGA GAT TTT CAG     7346 
Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln 
        385                 390                 395 

ATT ACA GCT TCA GGA CAA TAT GGA CAG TGG GCC CCA AAG CTG GCC AGA     7394 
Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg 
    400                 405                 410 

CTT CAT TAT TCC GGA TCA ATC AAT GCC TGG AGC ACC AAG GAG CCC TTT     7442 
Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe 
415                 420                 425                 430 

TCT TGG ATC AAG GTG GAT CTG TTG GCA CCA ATG ATT ATT CAC GGC ATC     7490 
Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile 
                435                 440                 445 

AAG ACC CAG GGT GCC CGT CAG AAG TTC TCC AGC CTC TAC ATC TCT CAG     7538 
Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln 
            450                 455                 460 

TTT ATC ATC ATG TAT AGT CTT GAT GGG AAG AAG TGG CAG ACT TAT CGA     7586 
Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg 
        465                 470                 475 

GGA AAT TCC ACT GGA ACC TTA ATG GTC TTC TTT GGC AAT GTG GAT TCA     7634 
Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser 
    480                 485                 490 

TCT GGG ATA AAA CAC AAT ATT TTT AAC CCT CCA ATT ATT GCT CGA TAC     7682 
Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 
495                 500                 505                 510 

ATC CGT TTG CAC CCA ACT CAT TAT AGC ATT CGC AGC ACT CTT CGC ATG     7730 
Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met 
                515                 520                 525 

GAG TTG ATG GGC TGT GAT TTA AAT AGT TGC AGC ATG CCA TTG GGA ATG     7778 
Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met 
            530                 535                 540 

GAG AGT AAA GCA ATA TCA GAT GCA CAG ATT ACT GCT TCA TCC TAC TTT     7826 
Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe 
        545                 550                 555 

ACC AAT ATG TTT GCC ACC TGG TCT CCT TCA AAA GCT CGA CTT CAC CTC     7874 
Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu 
    560                 565                 570 

CAA GGG AGG AGT AAT GCC TGG AGA CCT CAG GTG AAT AAT CCA AAA GAG     7922 
Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 
575                 580                 585                 590 

TGG CTG CAA GTG GAC TTC CAG AAG ACA ATG AAA GTC ACA GGA GTA ACT     7970 
Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr 
                595                 600                 605 

ACT CAG GGA GTA AAA TCT CTG CTT ACC AGC ATG TAT GTG AAG GAG TTC     8018 
Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe 
            610                 615                 620 

CTC ATC TCC AGC AGT CAA GAT GGC CAT CAG TGG ACT CTC TTT TTT CAG     8066 
Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln 
        625                 630                 635 

AAT GGC AAA GTA AAG GTT TTT CAG GGA AAT CAA GAC TCC TTC ACA CCT     8114 
Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro 
    640                 645                 650 

GTG GTG AAC TCT CTA GAC CCA CCG TTA CTG ACT CGC TAC CTT CGA ATT     8162 
Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile 
655                 660                 665                 670 

CAC CCC CAG AGT TGG GTG CAC CAG ATT GCC CTG AGG ATG GAG GTT CTG     8210 
His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu 
                675                 680                 685 

GGC TGC GAG GCA CAG GAC CTC TAC TGAGGGTGGC CACTGCAGCA CCTGCCACT     8264 
Gly Cys Glu Ala Gln Asp Leu Tyr 
            690 

CCGTCACCTC TCCCTCCTCA GCTCCAGGGC AGTGTCCCTC CCTGGCTTGC CTTCTACC     8324 

TGTGCTAAAT CCTAGCAGAC ACTGCCTTGA AGCCTCCTGA ATTAACTATC ATCAGTCC     8384 

CATTTCTTTG GTGGGGGGCC AGGAGGGTGC ATCCAATTTA ACTTAACTCT TACCTATT     8444 

CTGCAGCTGC TCCCAGATTA CTCCTTCCTT CCAATATAAC TAGGCAAAAA GAAGTGAG     8504 

GAAACCTGCA TGAAAGCATT CTTCCCTGAA AAGTTAGGCC TCTCAGAGTC ACCACTTC     8564 

CTGTTGTAGA AAAACTATGT GATGAAACTT TGAAAAAGAT ATTTATGATG TTAACTTG     8624 

TATTGCAGCT TATAATGGTT ACAAATAAAG CAATAGCATC ACAAATTTCA CAAATAAA     8684 

ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC ATCAATGTAT CTTATCAT     8744 

CTGGATCCCC GGGTGGCATC CCTGTGACCC CTCCCCAGTG CCTCTCCTGG CCCTGGAA     8804 

TGCCACTCCA GTGCCCACCA GCCTTGTCCT AATAAAATTA AGTTGCATCA TTTTGTCT     8864 

CTAGGTGTCC TTCTATAATA TTATGGGGTG GAGGGGGGTG GTATGGAGCA AGGGGCAA     8924 

TGGGAAGACA ACCTGTAGGG CCTGCGGGGT CTATTCGGGA ACCAAGCTGG AGTGCAGT     8984 

CACAATCTTG GCTCACTGCA ATCTCCGCCT CCTGGGTTCA AGCGATTCTC CTGCCTCA     9044 

CTCCCGAGTT GTTGGGATTC CAGGCATGCA TGACCAGGCT CAGCTAATTT TTGTTTTT     9104 

GGTAGAGACG GGGTTTCACC ATATTGGCCA GGCTGGTCTC CAACTCCTAA TCTCAGGT     9164 

TCTACCCACC TTGGCCTCCC AAATTGCTGG GATTACAGGC GTGAACCACT GCTCCCTT     9224 

CTGTCCTTCT GATTTTAAAA TAACTATACC AGCAGGAGGA CGTCCAGACA CAGCATAG     9284 

TACCTGCCAT GCCCAACCGG TGGGACATTT GAGTTGCTTG CTTGGCACTG TCCTCTCA     9344 

CGTTGGGTCC ACTCAGTAGA TGCCTGTTGA ATTCGTAATC ATGGTCATAG CTGTTTCC     9404 

TGTGAAATTG TTATCCGCTC ACAATTCCAC ACAACATACG AGCCGGAAGC ATAAAGTG     9464 

AAGCCTGGGG TGCCTAATGA GTGAGCTAAC TCACATTAAT TGCGTTGCGC TCACTGCC     9524 

CTTTCCAGTC GGGAAACCTG TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGG     9584 

GAGGCGGTTT GCGTATTGGG CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTC     9644 

TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCAC     9704 

AATCAGGGGA TAACGCAGGA AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAA     9764 

GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCA     9824 

AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGC     9884 

TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATA     9944 

TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGT     10004 

TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTC     10064 

CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACG     10124 

TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCG     10184 

CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGGACA GTATTTG     10244 

TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCG     10304 

AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCA     10364 

AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT CAGTGGA     10424 

AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA AAGGATCTTC ACCTAGA     10484 

TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAGTAT ATATGAGTAA ACTTGGT     10544 

ACAGTTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA TTTCGTT     10604 

CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC TTACCAT     10664 

GCCCCAGTGC TGCAATGATA CCGCGAGACC CACGCTCACC GGCTCCAGAT TTATCAG     10724 

TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA TCCGCCT     10784 

TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT AATAGTT     10844 

GCAACGTTGT TGCCATTGCT ACAGGCATCG TGGTGTCACG CTCGTCGTTT GGTATGG     10904 

CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG TTGTGCA     10964 

AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC GCAGTGT     11024 

CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC GTAAGAT     11084 

TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG CGGCGAC     11144 

GTTGCTCTTG CCCGGCGTCA ATACGGGATA ATACCGCGCC ACATAGCAGA ACTTTAA     11204 

TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA CCGCTGT     11264 

GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT TTTACTT     11324 

CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG GGAATAA     11384 

CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTTTCA ATATTATTGA AGCATTT     11444 

AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT AAACAAA     11504 

GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGACGT CTAAGAAACC ATTATTA     11564 

TGACATTAAC CTATAAAAAT AGGCGTATCA CGAGGCCCTT TCGTCTCGCG CGTTTCG     11624 

ATGACGGTGA AAACCTCTGA CACATGCAGC TCCCGGAGAC GGTCACAGCT TGTCTGT     11684 

CGGATGCCGG GAGCAGACAA GCCCGTCAGG GCGCGTCAGC GGGTGTTGGC GGGTGTC     11744 

GCTGGCTTAA CTATGCGGCA TCAGAGCAGA TTGTACTGAG AGTGCACCAT ATGCGGT     11804 

AAATACCGCA CAGATGCGTA AGGAGAAAAT ACCGCATCAG GCGCCATTCG CCATTCA     11864 

TGCGCAACTG TTGGGAAGGG CGATCGGTGC GGGCCTCTTC GCTATTACGC CAGCTGG     11924 

AAGGGGGATG TGCTGCAAGG CGATTAAGTT GGGTAACGCC AGGGTTTTCC CAGTCAC     11984 

GTTGTAAAAC GACGGCCAGT GCCAAGCTTG GGCTGCAG                          12022 

 
           
           
             
               11846 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
             
               CDS  
                1006..8058 

 
             
              4 

GTCGACGGTA TCGATAAGCT TGATATCGAA TTCCTGCAGC CCGGGGGATC CACTAGTACT     60 

CGAGACCTAG GAGTTAATTT TTAAAAAGCA GTCAAAAGTC CAAGTGGCCC TTGCGAGCA     120 

TTACTCTCTC TGTTTGCTCT GGTTAATAAT CTCAGGAGCA CAAACATTCC TTACTAGTC     180 

TAGAAGTTAA TTTTTAAAAA GCAGTCAAAA GTCCAAGTGG CCCTTGCGAG CATTTACTC     240 

CTCTGTTTGC TCTGGTTAAT AATCTCAGGA GCACAAACAT TCCTTACTAG TTCTAGAGC     300 

GCCGCCAGTG TGCTGGAATT CGGCTTTTTT AGGGCTGGAA GCTACCTTTG ACATCATTT     360 

CTCTGCGAAT GCATGTATAA TTTCTACAGA ACCTATTAGA AAGGATCACC CAGCCTCTG     420 

TTTTGTACAA CTTTCCCTTA AAAAACTGCC AATTCCACTG CTGTTTGGCC CAATAGTGA     480 

AACTTTTTCC TGCTGCCTCT TGGTGCTTTT GCCTATGGCC CCTATTCTGC CTGCTGAAG     540 

CACTCTTGCC AGCATGGACT TAAACCCCTC CAGCTCTGAC AATCCTCTTT CTCTTTTGT     600 

TTACATGAAG GGTCTGGCAG CCAAAGCAAT CACTCAAAGT TCAAACCTTA TCATTTTTT     660 

CTTTGTTCCT CTTGGCCTTG GTTTTGTACA TCAGCTTTGA AAATACCATC CCAGGGTTA     720 

TGCTGGGGTT AATTTATAAC TAAGAGTGCT CTAGTTTTGC AATACAGGAC ATGCTATAA     780 

AATGGAAAGA TGTTGCTTTC TGAGAGATCT CGAGGAAGCT AACAACAAAG AACAACAAA     840 

AACAATCAGG TAAGTATCCT TTTTACAGCA CAACTTAATG AGACAGATAG AAACTGGTC     900 

TGTAGAAACA GAGTAGTCGC CTGCTTTTCT GCCAGGTGCT GACTTCTCTC CCCTTCTCT     960 

TTTTCCTTTT CTCAGGATAA CAAGAACGAA ACAATAACAG CCACC ATG GAA ATA       1014 
                                                  Met Glu Ile 
                                                    1 

GAG CTC TCC ACC TGC TTC TTT CTG TGC CTT TTG CGA TTC TGC TTT AGT     1062 
Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe Cys Phe Ser 
      5                  10                  15 

GCC ACC AGA AGA TAC TAC CTG GGT GCA GTG GAA CTG TCA TGG GAC TAT     1110 
Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 
 20                  25                  30                  35 

ATG CAA AGT GAT CTC GGT GAG CTG CCT GTG GAC GCA AGA TTT CCT CCT     1158 
Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 
                 40                  45                  50 

AGA GTG CCA AAA TCT TTT CCA TTC AAC ACC TCA GTC GTG TAC AAA AAG     1206 
Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 
             55                  60                  65 

ACT CTG TTT GTA GAA TTC ACG GTT CAC CTT TTC AAC ATC GCT AAG CCA     1254 
Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro 
         70                  75                  80 

AGG CCA CCC TGG ATG GGT CTG CTA GGT CCT ACC ATC CAG GCT GAG GTT     1302 
Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val 
     85                  90                  95 

TAT GAT ACA GTG GTC ATT ACA CTT AAG AAC ATG GCT TCC CAT CCT GTC     1350 
Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 
100                 105                 110                 115 

AGT CTT CAT GCT GTT GGT GTA TCC TAC TGG AAA GCT TCT GAG GGA GCT     1398 
Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 
                120                 125                 130 

GAA TAT GAT GAT CAG ACC AGT CAA AGG GAG AAA GAA GAT GAT AAA GTC     1446 
Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 
            135                 140                 145 

TTC CCT GGT GGA AGC CAT ACA TAT GTC TGG CAG GTC CTG AAA GAG AAT     1494 
Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 
        150                 155                 160 

GGT CCA ATG GCC TCT GAC CCA CTG TGC CTT ACC TAC TCA TAT CTT TCT     1542 
Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 
    165                 170                 175 

CAT GTG GAC CTG GTA AAA GAC TTG AAT TCA GGC CTC ATT GGA GCC CTA     1590 
His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 
180                 185                 190                 195 

CTA GTA TGT AGA GAA GGG AGT CTG GCC AAG GAA AAG ACA CAG ACC TTG     1638 
Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 
                200                 205                 210 

CAC AAA TTT ATA CTA CTT TTT GCT GTA TTT GAT GAA GGG AAA AGT TGG     1686 
His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 
            215                 220                 225 

CAC TCA GAA ACA AAG AAC TCC TTG ATG CAG GAT AGG GAT GCT GCA TCT     1734 
His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 
        230                 235                 240 

GCT CGG GCC TGG CCT AAA ATG CAC ACA GTC AAT GGT TAT GTA AAC AGG     1782 
Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg 
    245                 250                 255 

TCT CTG CCA GGT CTG ATT GGA TGC CAC AGG AAA TCA GTC TAT TGG CAT     1830 
Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 
260                 265                 270                 275 

GTG ATT GGA ATG GGC ACC ACT CCT GAA GTG CAC TCA ATA TTC CTC GAA     1878 
Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 
                280                 285                 290 

GGT CAC ACA TTT CTT GTG AGG AAC CAT CGC CAG GCG TCC TTG GAA ATC     1926 
Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 
            295                 300                 305 

TCG CCA ATA ACT TTC CTT ACT GCT CAA ACA CTC TTG ATG GAC CTT GGA     1974 
Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 
        310                 315                 320 

CAG TTT CTA CTG TTT TGT CAT ATC TCT TCC CAC CAA CAT GAT GGC ATG     2022 
Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met 
    325                 330                 335 

GAA GCT TAT GTC AAA GTA GAC AGC TGT CCA GAG GAA CCC CAA CTA CGA     2070 
Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 
340                 345                 350                 355 

ATG AAA AAT AAT GAA GAA GCG GAA GAC TAT GAT GAT GAT CTT ACT GAT     2118 
Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 
                360                 365                 370 

TCT GAA ATG GAT GTG GTC AGG TTT GAT GAT GAC AAC TCT CCT TCC TTT     2166 
Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 
            375                 380                 385 

ATC CAA ATT CGC TCA GTT GCC AAG AAG CAT CCT AAA ACT TGG GTA CAT     2214 
Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 
        390                 395                 400 

TAC ATT GCT GCT GAA GAG GAG GAC TGG GAC TAT GCT CCC TTA GTC CTC     2262 
Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 
    405                 410                 415 

GCC CCC GAT GAC AGA AGT TAT AAA AGT CAA TAT TTG AAC AAT GGC CCT     2310 
Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 
420                 425                 430                 435 

CAG CGG ATT GGT AGG AAG TAC AAA AAA GTC CGA TTT ATG GCA TAC ACA     2358 
Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 
                440                 445                 450 

GAT GAA ACC TTT AAG ACT CGT GAA GCT ATT CAG CAT GAA TCA GGA ATC     2406 
Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 
            455                 460                 465 

TTG GGA CCT TTA CTT TAT GGG GAA GTT GGA GAC ACA CTG TTG ATT ATA     2454 
Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 
        470                 475                 480 

TTT AAG AAT CAA GCA AGC AGA CCA TAT AAC ATC TAC CCT CAC GGA ATC     2502 
Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 
    485                 490                 495 

ACT GAT GTC CGT CCT TTG TAT TCA AGG AGA TTA CCA AAA GGT GTA AAA     2550 
Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 
500                 505                 510                 515 

CAT TTG AAG GAT TTT CCA ATT CTG CCA GGA GAA ATA TTC AAA TAT AAA     2598 
His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 
                520                 525                 530 

TGG ACA GTG ACT GTA GAA GAT GGG CCA ACT AAA TCA GAT CCT CGG TGC     2646 
Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 
            535                 540                 545 

CTG ACC CGC TAT TAC TCT AGT TTC GTT AAT ATG GAG AGA GAT CTA GCT     2694 
Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 
        550                 555                 560 

TCA GGA CTC ATT GGC CCT CTC CTC ATC TGC TAC AAA GAA TCT GTA GAT     2742 
Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp 
    565                 570                 575 

CAA AGA GGA AAC CAG ATA ATG TCA GAC AAG AGG AAT GTC ATC CTG TTT     2790 
Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 
580                 585                 590                 595 

TCT GTA TTT GAT GAG AAC CGA AGC TGG TAC CTC ACA GAG AAT ATA CAA     2838 
Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 
                600                 605                 610 

CGC TTT CTC CCC AAT CCA GCT GGA GTG CAG CTT GAG GAT CCA GAG TTC     2886 
Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 
            615                 620                 625 

CAA GCC TCC AAC ATC ATG CAC AGC ATC AAT GGC TAT GTT TTT GAT AGT     2934 
Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 
        630                 635                 640 

TTG CAG TTG TCA GTT TGT TTG CAT GAG GTG GCA TAC TGG TAC ATT CTA     2982 
Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 
    645                 650                 655 

AGC ATT GGA GCA CAG ACT GAC TTC CTT TCT GTC TTC TTC TCT GGA TAT     3030 
Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 
660                 665                 670                 675 

ACC TTC AAA CAC AAA ATG GTC TAT GAA GAC ACA CTC ACC CTA TTC CCA     3078 
Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 
                680                 685                 690 

TTC TCA GGA GAA ACT GTC TTC ATG TCG ATG GAA AAC CCA GGT CTA TGG     3126 
Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 
            695                 700                 705 

ATT CTG GGG TGC CAC AAC TCA GAC TTT CGG AAC AGA GGC ATG ACC GCC     3174 
Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 
        710                 715                 720 

TTA CTG AAG GTT TCT AGT TGT GAC AAG AAC ACT GGT GAT TAT TAC GAG     3222 
Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 
    725                 730                 735 

GAC AGT TAT GAA GAT ATT TCA GCA TAC TTG CTG AGT AAA AAC AAT GCC     3270 
Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 
740                 745                 750                 755 

ATT GAA CCA AGA AGC TTC TCC CAG AAT TCA AGA CAC CCT AGC ACT AGG     3318 
Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg 
                760                 765                 770 

CAA AAG CAA TTT AAT GCC ACC ACA ATT CCA GAA AAT GAC ATA GAG AAG     3366 
Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys 
            775                 780                 785 

ACT GAC CCT TGG TTT GCA CAC AGA ACA CCT ATG CCT AAA ATA CAA AAT     3414 
Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn 
        790                 795                 800 

GTC TCC TCT AGT GAT TTG TTG ATG CTC TTG CGA CAG AGT CCT ACT CCA     3462 
Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro 
    805                 810                 815 

CAT GGG CTA TCC TTA TCT GAT CTC CAA GAA GCC AAA TAT GAG ACT TTT     3510 
His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe 
820                 825                 830                 835 

TCT GAT GAT CCA TCA CCT GGA GCA ATA GAC AGT AAT AAC AGC CTG TCT     3558 
Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser 
                840                 845                 850 

GAA ATG ACA CAC TTC AGG CCA CAG CTC CAT CAC AGT GGG GAC ATG GTA     3606 
Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val 
            855                 860                 865 

TTT ACC CCT GAG TCA GGC CTC CAA TTA AGA TTA AAT GAG AAA CTG GGG     3654 
Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly 
        870                 875                 880 

ACA ACT GCA GCA ACA GAG TTG AAG AAA CTT GAT TTC AAA GTT TCT AGT     3702 
Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser 
    885                 890                 895 

ACA TCA AAT AAT CTG ATT TCA ACA ATT CCA TCA GAC AAT TTG GCA GCA     3750 
Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala 
900                 905                 910                 915 

GGT ACT GAT AAT ACA AGT TCC TTA GGA CCC CCA AGT ATG CCA GTT CAT     3798 
Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His 
                920                 925                 930 

TAT GAT AGT CAA TTA GAT ACC ACT CTA TTT GGC AAA AAG TCA TCT CCC     3846 
Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 
            935                 940                 945 

CTT ACT GAG TCT GGT GGA CCT CTG AGC TTG AGT GAA GAA AAT AAT GAT     3894 
Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 
        950                 955                 960 

TCA AAG TTG TTA GAA TCA GGT TTA ATG AAT AGC CAA GAA AGT TCA TGG     3942 
Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp 
    965                 970                 975 

GGA AAA AAT GTA TCG TCA ACA GAG AGT GGT AGG TTA TTT AAA GGG AAA     3990 
Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys 
980                 985                 990                 995 

AGA GCT CAT GGA CCT GCT TTG TTG ACT AAA GAT AAT GCC TTA TTC AAA     4038 
Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 
                1000                1005                1010 

GTT AGC ATC TCT TTG TTA AAG ACA AAC AAA ACT TCC AAT AAT TCA GCA     4086 
Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 
            1015                1020                1025 

ACT AAT AGA AAG ACT CAC ATT GAT GGC CCA TCA TTA TTA ATT GAG AAT     4134 
Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu Asn 
        1030                1035                1040 

AGT CCA TCA GTC TGG CAA AAT ATA TTA GAA AGT GAC ACT GAG TTT AAA     4182 
Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu Phe Lys 
    1045                1050                1055 

AAA GTG ACA CCT TTG ATT CAT GAC AGA ATG CTT ATG GAC AAA AAT GCT     4230 
Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp Lys Asn Ala 
1060                1065                1070                1075 

ACA GCT TTG AGG CTA AAT CAT ATG TCA AAT AAA ACT ACT TCA TCA AAA     4278 
Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr Thr Ser Ser Lys 
                1080                1085                1090 

AAC ATG GAA ATG GTC CAA CAG AAA AAA GAG GGC CCC ATT CCA CCA GAT     4326 
Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly Pro Ile Pro Pro Asp 
            1095                1100                1105 

GCA CAA AAT CCA GAT ATG TCG TTC TTT AAG ATG CTA TTC TTG CCA GAA     4374 
Ala Gln Asn Pro Asp Met Ser Phe Phe Lys Met Leu Phe Leu Pro Glu 
        1110                1115                1120 

TCA GCA AGG TGG ATA CAA AGG ACT CAT GGA AAG AAC TCT CTG AAC TCT     4422 
Ser Ala Arg Trp Ile Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser 
    1125                1130                1135 

GGG CAA GGC CCC AGT CCA AAG CAA TTA GTA TCC TTA GGA CCA GAA AAA     4470 
Gly Gln Gly Pro Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys 
1140                1145                1150                1155 

TCT GTG GAA GGT CAG AAT TTC TTG TCT GAG AAA AAC AAA GTG GTA GTA     4518 
Ser Val Glu Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val 
                1160                1165                1170 

GGA AAG GGT GAA TTT ACA AAG GAC GTA GGA CTC AAA GAG ATG GTT TTT     4566 
Gly Lys Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe 
            1175                1180                1185 

CCA AGC AGC AGA AAC CTA TTT CTT ACT AAC TTG GAT AAT TTA CAT GAA     4614 
Pro Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu 
        1190                1195                1200 

AAT AAT ACA CAC AAT CAA GAA AAA AAA ATT CAG GAA GAA ATA GAA AAG     4662 
Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys 
    1205                1210                1215 

AAG GAA ACA TTA ATC CAA GAG AAT GTA GTT TTG CCT CAG ATA CAT ACA     4710 
Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr 
1220                1225                1230                1235 

GTG ACT GGC ACT AAG AAT TTC ATG AAG AAC CTT TTC TTA CTG AGC ACT     4758 
Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr 
                1240                1245                1250 

AGG CAA AAT GTA GAA GGT TCA TAT GAG GGG GCA TAT GCT CCA GTA CTT     4806 
Arg Gln Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu 
            1255                1260                1265 

CAA GAT TTT AGG TCA TTA AAT GAT TCA ACA AAT AGA ACA AAG AAA CAC     4854 
Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys His 
        1270                1275                1280 

ACA GCT CAT TTC TCA AAA AAA GGG GAG GAA GAA AAC TTG GAA GGC TTG     4902 
Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu Gly Leu 
    1285                1290                1295 

GGA AAT CAA ACC AAG CAA ATT GTA GAG AAA TAT GCA TGC ACC ACA AGG     4950 
Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys Thr Thr Arg 
1300                1305                1310                1315 

ATA TCT CCT AAT ACA AGC CAG CAG AAT TTT GTC ACG CAA CGT AGT AAG     4998 
Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr Gln Arg Ser Lys 
                1320                1325                1330 

AGA GCT TTG AAA CAA TTC AGA CTC CCA CTA GAA GAA ACA GAA CTT GAA     5046 
Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu 
            1335                1340                1345 

AAA AGG ATA ATT GTG GAT GAC ACC TCA ACC CAG TGG TCC AAA AAC ATG     5094 
Lys Arg Ile Ile Val Asp Asp Thr Ser Thr Gln Trp Ser Lys Asn Met 
        1350                1355                1360 

AAA CAT TTG ACC CCG AGC ACC CTC ACA CAG ATA GAC TAC AAT GAG AAG     5142 
Lys His Leu Thr Pro Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys 
    1365                1370                1375 

GAG AAA GGG GCC ATT ACT CAG TCT CCC TTA TCA GAT TGC CTT ACG AGG     5190 
Glu Lys Gly Ala Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg 
1380                1385                1390                1395 

AGT CAT AGC ATC CCT CAA GCA AAT AGA TCT CCA TTA CCC ATT GCA AAG     5238 
Ser His Ser Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys 
                1400                1405                1410 

GTA TCA TCA TTT CCA TCT ATT AGA CCT ATA TAT CTG ACC AGG GTC CTA     5286 
Val Ser Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu 
            1415                1420                1425 

TTC CAA GAC AAC TCT TCT CAT CTT CCA GCA GCA TCT TAT AGA AAG AAA     5334 
Phe Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys 
        1430                1435                1440 

GAT TCT GGG GTC CAA GAA AGC AGT CAT TTC TTA CAA GGA GCC AAA AAA     5382 
Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys 
    1445                1450                1455 

AAT AAC CTT TCT TTA GCC ATT CTA ACC TTG GAG ATG ACT GGT GAT CAA     5430 
Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln 
1460                1465                1470                1475 

AGA GAG GTT GGC TCC CTG GGG ACA AGT GCC ACA AAT TCA GTC ACA TAC     5478 
Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr 
                1480                1485                1490 

AAG AAA GTT GAG AAC ACT GTT CTC CCG AAA CCA GAC TTG CCC AAA ACA     5526 
Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 
            1495                1500                1505 

TCT GGC AAA GTT GAA TTG CTT CCA AAA GTT CAC ATT TAT CAG AAG GAC     5574 
Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys Asp 
        1510                1515                1520 

CTA TTC CCT ACG GAA ACT AGC AAT GGG TCT CCT GGC CAT CTG GAT CTC     5622 
Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu Asp Leu 
    1525                1530                1535 

GTG GAA GGG AGC CTT CTT CAG GGA ACA GAG GGA GCG ATT AAG TGG AAT     5670 
Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile Lys Trp Asn 
1540                1545                1550                1555 

GAA GCA AAC AGA CCT GGA AAA GTT CCC TTT CTG AGA GTA GCA ACA GAA     5718 
Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg Val Ala Thr Glu 
                1560                1565                1570 

AGC TCT GCA AAG ACT CCC TCC AAG CTA TTG GAT CCT CTT GCT TGG GAT     5766 
Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu Ala Trp Asp 
            1575                1580                1585 

AAC CAC TAT GGT ACT CAG ATA CCA AAA GAA GAG TGG AAA TCC CAA GAG     5814 
Asn His Tyr Gly Thr Gln Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu 
        1590                1595                1600 

AAG TCA CCA GAA AAA ACA GCT TTT AAG AAA AAG GAT ACC ATT TTG TCC     5862 
Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser 
    1605                1610                1615 

CTG AAC GCT TGT GAA AGC AAT CAT GCA ATA GCA GCA ATA AAT GAG GGA     5910 
Leu Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly 
1620                1625                1630                1635 

CAA AAT AAG CCC GAA ATA GAA GTC ACC TGG GCA AAG CAA GGT AGG ACT     5958 
Gln Asn Lys Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr 
                1640                1645                1650 

GAA AGG CTG TGC TCT CAA AAC CCA CCA GTC TTG AAA CGC CAT CAA CGG     6006 
Glu Arg Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg 
            1655                1660                1665 

GAA ATA ACT CGT ACT ACT CTT CAG TCA GAT CAA GAG GAA ATT GAC TAT     6054 
Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr 
        1670                1675                1680 

GAT GAT ACC ATA TCA GTT GAA ATG AAG AAG GAA GAT TTT GAC ATT TAT     6102 
Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr 
    1685                1690                1695 

GAT GAG GAT GAA AAT CAG AGC CCC CGC AGC TTT CAA AAG AAA ACA CGA     6150 
Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg 
1700                1705                1710                1715 

CAC TAT TTT ATT GCT GCA GTG GAG AGG CTC TGG GAT TAT GGG ATG AGT     6198 
His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser 
                1720                1725                1730 

AGC TCC CCA CAT GTT CTA AGA AAC AGG GCT CAG AGT GGC AGT GTC CCT     6246 
Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 
            1735                1740                1745 

CAG TTC AAG AAA GTT GTT TTC CAG GAA TTT ACT GAT GGC TCC TTT ACT     6294 
Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr 
        1750                1755                1760 

CAG CCC TTA TAC CGT GGA GAA CTA AAT GAA CAT TTG GGA CTC CTG GGG     6342 
Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly 
    1765                1770                1775 

CCA TAT ATA AGA GCA GAA GTT GAA GAT AAT ATC ATG GTA ACT TTC AGA     6390 
Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg 
1780                1785                1790                1795 

AAT CAG GCC TCT CGT CCC TAT TCC TTC TAT TCT AGC CTT ATT TCT TAT     6438 
Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr 
                1800                1805                1810 

GAG GAA GAT CAG AGG CAA GGA GCA GAA CCT AGA AAA AAC TTT GTC AAG     6486 
Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys 
            1815                1820                1825 

CCT AAT GAA ACC AAA ACT TAC TTT TGG AAA GTG CAA CAT CAT ATG GCA     6534 
Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala 
        1830                1835                1840 

CCC ACT AAA GAT GAG TTT GAC TGC AAA GCC TGG GCT TAT TTC TCT GAT     6582 
Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp 
    1845                1850                1855 

GTT GAC CTG GAA AAA GAT GTG CAC TCA GGC CTG ATT GGA CCC CTT CTG     6630 
Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu 
1860                1865                1870                1875 

GTC TGC CAC ACT AAC ACA CTG AAC CCT GCT CAT GGG AGA CAA GTG ACA     6678 
Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr 
                1880                1885                1890 

GTA CAG GAA TTT GCT CTG TTT TTC ACC ATC TTT GAT GAG ACC AAA AGC     6726 
Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser 
            1895                1900                1905 

TGG TAC TTC ACT GAA AAT ATG GAA AGA AAC TGC AGG GCT CCC TGC AAT     6774 
Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 
        1910                1915                1920 

ATC CAG ATG GAA GAT CCC ACT TTT AAA GAG AAT TAT CGC TTC CAT GCA     6822 
Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala 
    1925                1930                1935 

ATC AAT GGC TAC ATA ATG GAT ACA CTA CCT GGC TTA GTA ATG GCT CAG     6870 
Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln 
1940                1945                1950                1955 

GAT CAA AGG ATT CGA TGG TAT CTG CTC AGC ATG GGC AGC AAT GAA AAC     6918 
Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn 
                1960                1965                1970 

ATC CAT TCT ATT CAT TTC AGT GGA CAT GTG TTC ACT GTA CGA AAA AAA     6966 
Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 
            1975                1980                1985 

GAG GAG TAT AAA ATG GCA CTG TAC AAT CTC TAT CCA GGT GTT TTT GAG     7014 
Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu 
        1990                1995                2000 

ACA GTG GAA ATG TTA CCA TCC AAA GCT GGA ATT TGG CGG GTG GAA TGC     7062 
Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys 
    2005                2010                2015 

CTT ATT GGC GAG CAT CTA CAT GCT GGG ATG AGC ACA CTT TTT CTG GTG     7110 
Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val 
2020                2025                2030                2035 

TAC AGC AAT AAG TGT CAG ACT CCC CTG GGA ATG GCT TCT GGA CAC ATT     7158 
Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile 
                2040                2045                2050 

AGA GAT TTT CAG ATT ACA GCT TCA GGA CAA TAT GGA CAG TGG GCC CCA     7206 
Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 
            2055                2060                2065 

AAG CTG GCC AGA CTT CAT TAT TCC GGA TCA ATC AAT GCC TGG AGC ACC     7254 
Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr 
        2070                2075                2080 

AAG GAG CCC TTT TCT TGG ATC AAG GTG GAT CTG TTG GCA CCA ATG ATT     7302 
Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile 
    2085                2090                2095 

ATT CAC GGC ATC AAG ACC CAG GGT GCC CGT CAG AAG TTC TCC AGC CTC     7350 
Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu 
2100                2105                2110                2115 

TAC ATC TCT CAG TTT ATC ATC ATG TAT AGT CTT GAT GGG AAG AAG TGG     7398 
Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp 
                2120                2125                2130 

CAG ACT TAT CGA GGA AAT TCC ACT GGA ACC TTA ATG GTC TTC TTT GGC     7446 
Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly 
            2135                2140                2145 

AAT GTG GAT TCA TCT GGG ATA AAA CAC AAT ATT TTT AAC CCT CCA ATT     7494 
Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 
        2150                2155                2160 

ATT GCT CGA TAC ATC CGT TTG CAC CCA ACT CAT TAT AGC ATT CGC AGC     7542 
Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser 
    2165                2170                2175 

ACT CTT CGC ATG GAG TTG ATG GGC TGT GAT TTA AAT AGT TGC AGC ATG     7590 
Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met 
2180                2185                2190                2195 

CCA TTG GGA ATG GAG AGT AAA GCA ATA TCA GAT GCA CAG ATT ACT GCT     7638 
Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala 
                2200                2205                2210 

TCA TCC TAC TTT ACC AAT ATG TTT GCC ACC TGG TCT CCT TCA AAA GCT     7686 
Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 
            2215                2220                2225 

CGA CTT CAC CTC CAA GGG AGG AGT AAT GCC TGG AGA CCT CAG GTG AAT     7734 
Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn 
        2230                2235                2240 

AAT CCA AAA GAG TGG CTG CAA GTG GAC TTC CAG AAG ACA ATG AAA GTC     7782 
Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val 
    2245                2250                2255 

ACA GGA GTA ACT ACT CAG GGA GTA AAA TCT CTG CTT ACC AGC ATG TAT     7830 
Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr 
2260                2265                2270                2275 

GTG AAG GAG TTC CTC ATC TCC AGC AGT CAA GAT GGC CAT CAG TGG ACT     7878 
Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr 
                2280                2285                2290 

CTC TTT TTT CAG AAT GGC AAA GTA AAG GTT TTT CAG GGA AAT CAA GAC     7926 
Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 
            2295                2300                2305 

TCC TTC ACA CCT GTG GTG AAC TCT CTA GAC CCA CCG TTA CTG ACT CGC     7974 
Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg 
        2310                2315                2320 

TAC CTT CGA ATT CAC CCC CAG AGT TGG GTG CAC CAG ATT GCC CTG AGG     8022 
Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg 
    2325                2330                2335 

ATG GAG GTT CTG GGC TGC GAG GCA CAG GAC CTC TAC TGAGGGTGGC          8068 
Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 
2340                2345                2350 

CACTGCAGCA CCTGCCACTG CCGTCACCTC TCCCTCCTCA GCTCCAGGGC AGTGTCCC     8128 

CCTGGCTTGC CTTCTACCTT TGTGCTAAAT CCTAGCAGAC ACTGCCTTGA AGCCTCCT     8188 

ATTAACTATC ATCAGTCCTG CATTTCTTTG GTGGGGGGCC AGGAGGGTGC ATCCAATT     8248 

ACTTAACTCT TACCTATTTT CTGCAGCTGC TCCCAGATTA CTCCTTCCTT CCAATATA     8308 

TAGGCAAAAA GAAGTGAGGA GAAACCTGCA TGAAAGCATT CTTCCCTGAA AAGTTAGG     8368 

TCTCAGAGTC ACCACTTCCT CTGTTGTAGA AAAACTATGT GATGAAACTT TGAAAAAG     8428 

ATTTATGATG TTAACTTGTT TATTGCAGCT TATAATGGTT ACAAATAAAG CAATAGCA     8488 

ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAAC     8548 

ATCAATGTAT CTTATCATGT CTGGATCCCC GGGTGGCATC CCTGTGACCC CTCCCCAG     8608 

CCTCTCCTGG CCCTGGAAGT TGCCACTCCA GTGCCCACCA GCCTTGTCCT AATAAAAT     8668 

AGTTGCATCA TTTTGTCTGA CTAGGTGTCC TTCTATAATA TTATGGGGTG GAGGGGGG     8728 

GTATGGAGCA AGGGGCAAGT TGGGAAGACA ACCTGTAGGG CCTGCGGGGT CTATTCGG     8788 

ACCAAGCTGG AGTGCAGTGG CACAATCTTG GCTCACTGCA ATCTCCGCCT CCTGGGTT     8848 

AGCGATTCTC CTGCCTCAGC CTCCCGAGTT GTTGGGATTC CAGGCATGCA TGACCAGG     8908 

CAGCTAATTT TTGTTTTTTT GGTAGAGACG GGGTTTCACC ATATTGGCCA GGCTGGTC     8968 

CAACTCCTAA TCTCAGGTGA TCTACCCACC TTGGCCTCCC AAATTGCTGG GATTACAG     9028 

GTGAACCACT GCTCCCTTCC CTGTCCTTCT GATTTTAAAA TAACTATACC AGCAGGAG     9088 

CGTCCAGACA CAGCATAGGC TACCTGCCAT GCCCAACCGG TGGGACATTT GAGTTGCT     9148 

CTTGGCACTG TCCTCTCATG CGTTGGGTCC ACTCAGTAGA TGCCTGTTGA ATTCGTAA     9208 

ATGGTCATAG CTGTTTCCTG TGTGAAATTG TTATCCGCTC ACAATTCCAC ACAACATA     9268 

AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA GTGAGCTAAC TCACATTA     9328 

TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG TCGTGCCAGC TGCATTAA     9388 

AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCTCTTCCG CTTCCTCG     9448 

CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAG     9508 

GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT GAGCAAAA     9568 

CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTC     9628 

CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACA     9688 

ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCG     9748 

CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCT     9808 

TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGT     9868 

GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAG     9928 

CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGC     9988 

AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTA     10048 

TAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAG     10108 

TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTG     10168 

GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTAC     10228 

GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATC     10288 

AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAG     10348 

ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCAC CTATCTC     10408 

GATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTAC     10468 

ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGACC CACGCTC     10528 

GGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGG     10588 

TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAG     10648 

TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCATCG TGGTGTC     10708 

CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTAC     10768 

ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAG     10828 

TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTAC     10888 

CATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTG     10948 

ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATA ATACCGC     11008 

ACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACT     11068 

AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTG     11128 

TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAA     11188 

CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTT     11248 

ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATG     11308 

TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGA     11368 

CTAAGAAACC ATTATTATCA TGACATTAAC CTATAAAAAT AGGCGTATCA CGAGGCC     11428 

TCGTCTCGCG CGTTTCGGTG ATGACGGTGA AAACCTCTGA CACATGCAGC TCCCGGA     11488 

GGTCACAGCT TGTCTGTAAG CGGATGCCGG GAGCAGACAA GCCCGTCAGG GCGCGTC     11548 

GGGTGTTGGC GGGTGTCGGG GCTGGCTTAA CTATGCGGCA TCAGAGCAGA TTGTACT     11608 

AGTGCACCAT ATGCGGTGTG AAATACCGCA CAGATGCGTA AGGAGAAAAT ACCGCAT     11668 

GCGCCATTCG CCATTCAGGC TGCGCAACTG TTGGGAAGGG CGATCGGTGC GGGCCTC     11728 

GCTATTACGC CAGCTGGCGA AAGGGGGATG TGCTGCAAGG CGATTAAGTT GGGTAAC     11788 

AGGGTTTTCC CAGTCACGAC GTTGTAAAAC GACGGCCAGT GCCAAGCTTG GGCTGCA     11846 

 
           
           
             
               211 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              5 

ATTGAACCAA GAAGCTTCTC CCAGGTAAGT TGCTAATAAA GCTTGGCAAG AGTATTTCAA     60 

GGAAGATGAA GTCATTAACT ATGCAAAATG CTTCTCAGGC ACCTAGGAAA ATGAGGATG     120 

GAGGCATTTC TACCCACTTG GTACATAAAA TTATTGCTTT TCCTCTTCTT TTTTTCTCC     180 

GAACCCACCA GTCTTGAAAC GCCATCAACG G                                   211 

 
           
           
             
               126 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              6 

GTTGGTATCC TTTTTACAGC ACAACTTAAT GAGACAGATA GAAACTGGTC TTGTAGAAAC     60 

AGAGTAGTCG CCTGCTTTTC TGCCAGGTGC TGACTTCTCT CCCCTGGGCT GTTTTCATT     120 

TCTCAG                                                               126 

 
           
           
             
               126 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              7 

GTAAGTATCC TTTTTACAGC ACAACTTAAT GAGACAGATA GAAACTGGTC TTGTAGAAAC     60 

AGAGTAGTCG CCTGCTTTTC TGCCAGGTGC TGACTTCTCT CCCCTTCTCT TTTTTCCTT     120 

TCTCAG                                                               126 

 
           
           
             
               10 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              8 

GCCACCAUGG                                                            10 

 
           
           
             
               100 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              9 

AGGTTAATTT TTAAAAAGCA GTCAAAAGTC CAAGTGGCCC TTGCGAGCAT TTACTCTCTC     60 

TGTTTGCTCT GGTTAATAAT CTCAGGAGCA CAAACATTCC                          100 

 
           
           
             
               223 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              10 

CTTTCTCTTT TCTTTTACAT GAAGGGTCTG GCAGCCAAAG CAATCACTCA AAGTTCAAAC     60 

CTTATCATTT TTTGCTTTGT TCCTCTTGGC CTTGGTTTTG TACATCAGCT TTGAAAATA     120 

CATCCCAGGG TTAATGCTGG GGTTAATTTA TAACTAAGAG TGCTCTAGTT TTGCAATAC     180 

GGACATGCTA TAAAAATGGA AAGATGTTGC TTTCTGAGAG ATA                      223 

 
           
           
             
               90 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              11 

AGAUCUCGAG AAAGCUAACA ACAAAGAACA ACAAACAACA AUCAGGAUAA CAAGAACGAA     60 

ACAAUAACAG CCACCAUGGA AAUAGAGCUC                                      90