Patent Application: US-68864996-A

Abstract:
a pol - ii type dna polymerase wherein an alanine located at the nucleotide binding site is replaced with a hydroxy containing amino acid .

Description:
fig1 shows the amino acid sequence of wild type dna polymerase of pyrococcus furiosis ( seq . id . no . 37 ). fig2 shows the nucleic acid sequence of wild type dna polymerase of pyrococcus furiosis ( seq . id . no . 38 ). fig3 is a representation of a sequencing gel from reactions using wild type or a491y pfu polymerase . the following examples serve to illustrate the dna polymerases of the present invention and their use in sequencing . dna polymerase activity was assayed by measuring the amount of incorporation of a radiolabeled deoxynucleotide into acid precipitable material using activated salmon sperm dna as a template ( richardson , c . c . ( 1966 ) dna polymerase from escherichia coli , pp . 263 - 276 in g . l . cantoni and d . r . davies ( ed . ), procedures in nucleic acid research . harper and row , new york ). a unit of dna polymerase is the amount of enzyme that catalyzes incorporation of 10 nmoles of deoxynucleotide triphosphate into acid - precipitable material in 30 minutes at 70 ° c . an assay consists of 2 - 10 μl of protein solution containing dna polymerase being incubated with 50 μl of assay mix ( 20 mm tris hcl ph 8 . 5 @ room temperature , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 0 . 1 % ( v / v ) triton x - 100 , 100 μg / ml bsa , 2 mm mgso 4 , 200 μm each deoxynucleotide triphosphate , 0 . 2 mg / ml activated salmon sperm dna , 1 μci 3000 ci / mmole α - 33 p ! datp ) at 70 ° c . for 10 minutes . the reaction is stopped by adding the contents of the assay to a tube containing 1 ml each of carrier 50 μg / ml fish sperm dna , 2 mm edta ) and precipitant ( 20 % ( w / v ) trichloroacetic acid , 2 % ( w / v ) sodium pyrophosphate ). after incubation on ice for at least 5 minutes , the solution is filtered through a glass fiber filter ( e . g . gf / b , whatman ), washed with several mililiters ice - cold wash solution ( 1n hydrochloric acid , 100 mm sodium pyrophosphate ), placed in a counting vial with aqueous liquid scintillation cocktail ( e . g . biosafe ii , research products international corp .) and counted in a liquid scintillation counter . dna polymerase activity of the test solution is calculated from the measured specific activity of the assay mix . the assay has been shown to be linear between 0 . 01 to 0 . 2 units per assay reaction and only values between these were accepted as significant . solution protein concentrations are measured spectrophotometrically by determining the absorbance of the test solution at a wavelength of 205 nm ( a 205 ) ( scopes , r . k . ( 1994 ) pp . 46 - 48 protein purification . springer - verlag , new york ). the extinction coefficient of polypeptides is taken as e 205 ( 1 mg · ml - 1 )= 31 . exonuclease activity was measured as described ( kong , h ., kucera , r . b . and jack , w ., e . ( 1993 ) j . biol . chem . 268 , 1965 - 1975 ) using , as substrate a 500 bp pcr product generated from pbr322 labeled with 3 h by incorporation of tritiated ttp . exonuclease assays were performed in the same buffer ( 20 mm tris hcl ph 8 . 5 @ room temperature , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 0 . 1 % ( v / v ) triton x - 100 , 100 μg / ml bsa , 2 mm mgso 4 ) and at the same temperature ( 70 ° c .) as polymerase assays . it has been shown that overproduction by cloning of trna arg can significantly increase the expression of exogenous proteins in e . coli ( brinkmann et al ., ( 1989 ) gene 85 , 109 - 114 ; schenk et al . ( 1995 ) biotechniques 19 , 196 - 200 ). novel expression vectors were created by cloning the gene for trna arg onto a vector with tightly - regulated inducible expression for exogenous proteins such as dna polymerases . to construct these novel vectors , two pcr primers ggaattcagatctaaaagccattgactcagcaag ( seq . id . no . 1 ) and ggaattcacatgtgacagagcatgcgaggaaaat ( seq . id . no . 2 ) were designed to amplify the gene , promotor and terminator sequences of trna arg ( argu gene ) from e . coli genomic dna . the sequence of this gene and its regulatory regions has been previously published ( garcia et al . ( 1986 ) cell 45 , 453 - 459 ). e . coli genomic dna was prepared from strain hms 174 ( atcc # 47001 ). the pcr product was cloned using the vector provided as part of the pmos blue t - vector kit ( amersham life science ), and two clones were confirmed to have the correct sequence . the argu gene was excised from this vector by means of restriction digestion with the enzyme ecor i . this fragment was ligated to vector pre2 ( reddy et al . ( 1989 ) nucleic acids res . 17 , 10473 - 10488 ) which was digested with ecor i and dephosphorylated . resulting plasmids were screened for the presence of the argu gene , and inserts of both orientations were obtained . the new plasmid carrying the argu gene transcribed in the same direction as the vector gene for beta - lactamase is designated prd ( seq . id no . 3 ); the plasmid with argu transcribed in the opposite direction is designated prd2 ( seq . id . no . 4 ). a culture of pyrococcus furiosus was a gift of dr . frank t . robb . the culture was grown at 90 ° c . essentially as described ( archaea : a laboratory manual ( 1995 ) robb , f . t . ( ed . ), cold spring habor laboratory press , plainview , n . y .). the medium contained : per liter , 5 g tryptone , 1 g yeast extract , 24 g nacl , 4 g na 2 so 4 , 0 . 7 g kcl , 0 . 2 g nahco 3 , 0 . 1 g kbr , 30 mg h 3 bo 3 , 10 . 8 g mgcl 2 . 6h 2 o , 1 . 5 g cacl 2 , 25 mg srcl 2 , 0 . 4 mg resazurin . frozen cell paste of pyrococcus furiosus ( approximately 350 mg ) was resuspended in 700 μl of lysis buffer ( 50 mm tris . hcl ph 8 . 0 , 50 mm edta , 3 % sds ( w / v ), 1 % 2 - mercaptoethanol ( v / v )) and incubated for 1 hour at 65 ° c . this solution was extracted three times with 25 : 24 : 1 phenol : chloroform : isoamyl alcohol , twice with chloroform and precipitated with two volumes of 100 % ethanol . the pellet was dried briefly in vacuo and dissolved in 700 μl te ( 10 mm tris . hcl ph 8 . 0 , 1 mm edta ). the concentration of genomic dna was determined spectrophotometrically by measuring the absorbance at 260 nm ( 1a 260 = 50 μg / ml ) and by comparison of uv fluorescence of bands on an ethidium bromide stained agarose gel relative to standard concentration markers . pcr primers to amplify the dna polymerase gene from the genome of pyrococcus furiosus were designed from the published gene sequence ( uemori , t ., ishino , y ., toh , h ., asada , f . and kato , i . ( 1993 ) nucleic acids res . 21 , 259 - 265 ) ( seq . id . no . 38 ). the 5 &# 39 ; primer , pfupolf ( ggggtaccatatgattttagatgtggattacataac )( seq . id . no . 5 ) hybridizes to nucleotides 1 - 26 of the dna polymerase coding sequence and introduces a unique nde i ( catatg ) site on the amplified product . the 3 &# 39 ; primer , pfupolr2 ( tcccccgggctaggattttttaatgttaagcca )( seq . id . no . 6 ) hybridizes to nucleotides 2305 - 2328 of the dna polymerase coding sequence and introduces a unique sma i site ( cccggg ) on the amplified product . to minimize errors introduced by taq dna polymerase during pcr , a modified &# 34 ; long pcr &# 34 ; was carried out . the pcr reaction contained : 20 mm tricine ( ph 8 . 8 ), 85 mm koac , 200 μm each dntp , 5 % dmso , 0 . 5 mm each primer , 1 . 5 mm mgoac ( added last as hot - start ), 2 . 5 units hot tub dna polymerase per 100 μl reaction ( amersham ), 0 . 025 u deep vent dna polymerase per 100 μl reaction ( new england biolabs ) and 20 - 100 ng pyrococcus furiosus genomic dna per 100 μl reaction . the cycling parameters consisted of : 94 ° c . 30 seconds , then 68 ° c . 10 minutes 40 seconds × 8 cycles ; 94 ° c . 30 seconds , then 68 ° c . 12 minutes × 8 cycles ; 94 ° c . 30 seconds , then 68 ° c . 13 minutes 20 seconds × 8 cycles ; 94 ° c . 30 s , then 68 ° c . 14 minutes 40 seconds × 8 cycles . the resulting pcr product was digested with nde i and sma i and cloned into similarly digested pre2 ( reddy , p ., peterkofsky , a . and mckenney , k . ( 1989 ) nucleic acids res . 17 , 10473 - 10488 ) to produce pre2pfuwt . constructs made with pre based vectors were propagated in e . coli strain dh - 1 λ +( λcl +). the cloned gene was verified by dna sequencing . based on alignments of dna polymerases ( delarue , m ., poch , o ., tordo , n ., moras , d . and argos , p . ( 1990 ) protein engineering 3 , 461 - 467 ; braithwaite , d . k . and ito , j . ( 1993 ) nucleic acids res . 21 , 787 - 202 ), the aspartic acid at amino acid position 215 was identified as being homologous to asp66 of . o slashed . 29 dna polymerase . an acidic residue is highly conserved at this position among dna polymerases possessing 3 &# 39 ;- 5 &# 39 ; exonuclease activity . mutation of asp66 in . o slashed . 29 dna polymerase to alanine ( d66a ) resulted in a polymerase which had exonuclease activity reduced about 1000 - fold relative to the wild type enzyme without affecting dna polymerase activity ( bernard , a ., blanco , l ., lβzaro , j ., martin , g . and salas , m . ( 1989 ) cell 59 , 219 - 228 ). to accomplish the analogous mutation in the pyrococcus furiosus dna polymerase gene , two primers were made . primer d214abam ( aaggatcctgacattatagttacttataatggagactcattcgccttccc ) ( seq . id . no . 7 ) covers the bamh i site at nt 603 of the coding sequence and is mutagenic for the asp215 codon at nt 644 ( gac -& gt ; gcc ). primer d214aeco ( ggaattctttcccgagttcataag ) ( seq . id . no . 8 ) covers the ecor i site at nt 973 . these primers were used in pcr to simultaneously amplify the region between them and introduce the mutation . the resulting pcr product was digested with ecor i and bamh i and subcloned into pre2pfuwt to produce ptm100 . in order to favor high - level expression of the dna polymerase gene in e . coli , the 5 &# 39 ; end of the gene was modified in two ways . firstly , since the codon utilization of pyrococcus furiosus differs significantly from that of e . coli , the first 58 codons of the gene were changed to synonymous codons favored by e . coli for highly expressed genes . secondly , to further mimic a highly expressed e . coli coding sequence , an alanine codon ( gct ) was inserted after the initiating methionine . two long oligonucleotides , mod95f ( gctatcctggacgttgactacatcaccgaagaaggtaagccggttatccgtctgt tcaaaaaagaaaacggtaaattcaaaatcgaacacgaccg )( seq . id . no . 9 ) and mod95r ( accggtgatttttttaacttcttcgattttagagtcgtcacgcagcagagcgtag atgtacggacggaaggtacggtcgtgttcgattttgaatt )( seq . id . no . 10 ) which are antiparallel at their 3 &# 39 ; ends were used together with primers mod37f ( ggggtaccatatggctatcctggacgttgactacatc )( seq . id . no . 11 ) and mod32r ( ggggtaccaccggtgatttttttaacttcttc )( seq . id . no . 12 ) in a pcr reaction containing 17 . 5 nm each mod95f and mod95r and 500 nm each mod37f and mod32r . the resulting 189 bp product contained an nde i site corresponding to the start of the coding sequence , the changed codons discussed above and a silent age i site . the product was cloned into pmosblue pcr product cloning vector ( amersham ) to produce plasmid ptm101 and verified by sequencing . to introduce an age i site into the gene , pcr was carried out using the unmodified clone ( ptm100 ) as template with primers ageimut ( aaataaccggtgaacgtcatggaaagattgtg )( seq . id . no . 13 ) which introduces a silent mutation for the age i site and r467 ( cctttgcttcattttcatctg )( seq . id . no . 14 ). the resulting 350 bp pcr product was cloned into pmosblue to produce plasmid ptm102 . the modified gene was constructed by ligating the 165 bp fragment of ptm101 digested with nde i and age i , the 165 bp fragment of ptm102 digested with age i and bstb i and the & gt ; 5 kb fragment of pre2pfuwt ( for exo +) or ptm100 ( for exo -) digested with nde i and bstb i . the resulting exo + construct is designated ptm103 , the exo - construct ptm104 . constructs were verified by restriction digestion with each enzyme used in cloning . twelve cassette oligonucleotides , six for the sense strand and six for the antisense , which match the sequence of the region between the econ i ( nt 1309 of the modified coding sequence ) and sac i ( nt 1585 ) restriction sites except for silent mutations which introduce xho i and bssh ii sites at nts 1372 and 1507 , respectively , were constructed . the cassette oligos are : oligonucleotides were purchased 5 &# 39 ; phosphorylated using phosphalink amidite ( abi ). each oligonucleotide was made 20 μm in te ( 10 mm tris . hcl ph 8 . 0 , 0 . 1 mm edta ). in one reaction , 2 μl of each oligonucleotide were combined with 3 μl 10 × taq ligase buffer and 1 μl ( 40 units ) taq dna ligase ( new england biolabs ) and incubated 30 minutes at 45 ° c . pcr was carried out using 1 μl of this ligated material as substrate and primers caspcrf2 ( tcgctcctcaagtaggccacaagttctgcaaggacatccc )( seq . id . no . 27 ) and caspcrr2 ( tcttcgagctccttccatactaactcgatgtactttcttc )( seq . id . no . 28 ). the resulting product was digested with sac i and econi and cloned into similarly digested parent vector ptm103 to produce ptm119 or the same construct in prd to produce ptm118 . e . coli strain m5248 ( ci857 iysogen ) harboring the expression plasmid was cultured in lb medium ( per liter : 10 g tryptone , 5 g yeast extract , 10 g nacl ) supplemented with 50 μg / ml ampicillin . the culture was grown at 30 - 32 ° c . until the od 600 reached approximately 1 . 0 at which time the culture temperature was raised to 40 °- 42 ° c . growth continued at this temperature for 2 hours . the cells were harvested and frozen at - 20 ° c . until needed . frozen cell paste was resuspended in buffer a ( 50 mm tris . hcl ph 8 . 0 , 1 mm edta , 0 . 1 % ( v / v ) each , np40 and tween - 20 ) containing 100 mm nacl , 1 mm pmsf , lysozyme ( 0 . 2 mg · ml - 1 ) and dnase i ( 10 μg · ml - 1 ) at 0 . 1 ml buffer per ml original culture volume . after 15 minutes incubation at room temperature , the lysate was subjected to brief sonication , and cleared by centrifugation . the cleared lysate was heated to 70 ° c . for 10 - 15 minutes , incubated on ice for 10 minutes and centrifuged again . this heat treatment inactivates detectable dna polymerase activity resulting from the e . coli host polymerases . the cleared , heat - treated lysate is designated fraction i . fraction i was passed through a column of deae cellulose ( de - 52 , whatman ) equilibrated in buffer a containing 100 mm nacl and washed with the same buffer . the flow - through fractions containing dna polymerase activity were collected and loaded onto a column of heparin sepharose ( heparin sepharose cl - 6b , pharmacia ) equilibrated in the same buffer . the column was washed and the dna polymerase activity was eluted with a linear salt gradient of 100 - 700 mm nacl in buffer a with the peak of activity eluting at about 300 mm nacl . the active fractions were pooled , concentrated via ultrafiltration ( centricon - 50 , amicon ) and dialyzed against storage buffer ( 50 mm tris hcl ph 8 . 2 @ room temperature , 0 . 1 mm edta , 0 . 1 % ( v / v ) triton x - 100 , 0 . 1 % ( v / v ) np40 and 50 % ( v / v ) glycerol ). mutations were introduced into the coding sequence of the pyrococcus furiosus dna polymerase gene in a manner analogous to the cassette mutagenesis used to introduce the bsshii and xho i restriction sites . for example , mutant a491y was constructed as follows : oligonucleotides casst1 , casst2 , casst3 , casst5 , casst6 , cassb1 , cassb2 , cassb4 , cassb5 , cassb6 described above were combined with two mutagenic oligonucleotides , a491yt ( aaaactcttatacaattctttctacggatattatggctatgcaaaagcgc ) seq . id . no . 29 ) and a491yb ( gaaagaattgtataagagttttatcgctttttgtctatagtcaaggagta ) ( seq . id . no . 30 ), both 5 &# 39 ; phosphorylated as above to a final concentration of 1 . 7 μm each oligonucleotide in te ( 10 mm tris . hcl ph 8 . 0 , 0 . 1 mm edta ). these two primers are identical to casst4 and cassb3 , respectively , except the codon for alanine 491 ( gca ) has been changed to tyrosine ( tac ). to 48 μl of the oligonucleotide mix was add 8 . 5 μl of annealing buffer ( 50 mm mgcl 2 , 100 mm tris . hcl ph 7 . 5 and 300 mm nacl ). the mixture was heated to 100 ° c . and slow cooled to 50 ° c . at which time 8 μl was withdrawn and added to 1 μl taq dna ligase buffer and 1 μl ( 40 units ) taq dna ligase ( new england biolabs ). incubation continued for 30 minutes at 50 ° c . pcr was carried out with ultma dna polymerase ( perkin - elmer ) using 1 μl of this ligated material as template and oligonucleotides casst1 and cassb1 as pcr primers . the resulting 275 bp product was extracted with chcl 3 , digested with xho i and bssh ii , and cloned into similarly digested ptm118 to produce ptm113 . in a similar fashion , three other mutants were constructed . using oligonucleotides n492yt ( aaaactcttagcatactctttctacggatattatggctatgcaaaagcgc ) seq . id . no . 31 ) and n492yb ( gaaagagtatgctaagagttttatcgctttttgtctatagtcaaggagta ) seq . id . no . 32 ), a construct was made which has tyrosine substituted for asparagine 492 ( n492y ). using oligonucleotides 0491yt ( aaaactcttagcatacaattctttctacggatattatggctatgcaaaagcgc ) seq . id . no . 33 ) and 0491yb ( gaaagaattgtatgctaagagttttatcgctttttgtctatagtcaaggagta ) seq . id . no . 34 ), a construct was made which has tyrosine inserted between alanine 491 and asparagine 492 ( ω491y ). using oligonucleotides otaq7t ( aaaaaccatcaactacggtgttctctacggatattatggctatgcaaaagcgc ) seq . id . no . 35 ) and otaq7b ( gagaacaccgtagttgatggtttttatcgctttttgtctatagtcaaggagta ) seq . id . no . 36 ), a construct was made which has the sequence of seven amino acids tinygvl replacing the sequence of six amino acids llansf at position # 490 - 495 of the pyrococcus furiosus dna polymerase coding sequence ( ωtaq7 ). mutant dna polymerases were characterized by performing sequencing reactions . the reaction contained annealed primer : template ( 0 . 5 pmole universal cycle primer ( amersham ) labeled on the 5 &# 39 ; end by the action of t4 polynucleotide kinase and γ - 33 p ! atp annealed to 1 μg m13mp18 ) in reaction buffer ( 20 mm tris hcl ph 8 . 5 @ room temperature , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 0 . 1 % ( v / v ) triton x - 100 , 100 μg / ml bsa , 2 mm mgso 4 ) and 4 - 8 units of polymerase . this mixture was divided into four and added to tubes containing 4 μl termination mix . all termination mixes consisted of 50 μm each dntp in reaction buffer and various amounts of ddntp . a complement of termination mixes at 100 : 1 ddntp : dntp , for example , contained 5 mm ddg , dda , ddt or ddc in addition to the dntps and buffer . upon addition of the enzyme primer : template to the termination mixes , the reactions were incubated at 70 ° c . for 15 - 30 minutes , stopped by addition of 4 μl stop solution ( 95 % formamide , 10 mm edta , 0 . 1 % each bromophenol blue and xylene cyanol ), heated to ca . 75 ° c . for 5 minutes and loaded onto a denaturing polyacrylamide gel and electrophoresed under standard conditions . an autoradiogram of the electrophoresed gel was subjected to densitometry with an optical scanner ( sciscan , usb ) to determine band intensity . wild type pyrococcus furiosus dna polymerase requires termination mixes that are 100 : 1 ddntp : dntp to bring about chain termination events distributed evenly in the region 1 - 400 nt downstream of the primer . the root mean square (&# 34 ; rms value &# 34 ;) of the measured band intensities in the region about 50 - 200 nt downstream of the primer is approximately 0 . 65 . in other words , the standard deviation of the measured band intensities is 65 % of the mean value . the mutant enzyme a491y dna polymerase required termination mixes adjusted to 25 : 1 , 50 : 1 , 25 : 1 and 25 : 1 ddntp : dntp for g , a , t and c , respectively , to bring about chain termination events distributed evenly in the region 1 - 400 nt downstream of the primer . using the same ratios as wild type ( 100 : 1 ddntp : dntp ) resulted in very short extension products i . e . less than 40 from the primer . therefore , the a491y mutant is able to utilize ddntps more efficiently than the wild type enzyme . more significantly , the band intensities on the autoradiogram of an optimized reaction are much more uniform for the a491y mutant dna polymerase than for the wild type dna polymerase having and rms value of 0 . 3 . this rms value reflects a great improvement in band uniformity . fig3 shows sequencing data produced by wild type and a491y dna polymerases . the mutant enzymes ω491y ( tyrosine inserted between a491 and n492 ), ωtaq7 ( amino acids 489 - 494 replaced with 7 corresponding amino acids from taq polymerase including an f -& gt ; y substitution ) and n492y were purified and all had significantly reduced specific activities ( greater than 100 - fold of wild type ). __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 38 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 34 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 1 : ggaattcagatctaaaagccattgactcagcaag34 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 34 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 2 : ggaattcacatgtgacagagcatgcgaggaaaat34 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 5249 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 3 : ttcacatgtgacagagcatgcgaggaaaatagacgacatttttggtgataatgtcccaaa60tatgtcccactctgaaattatggaggatataaagaaggcgtaactgattgaattgtaatg120gcgcgccctgcaggattcgaacctgcggcccacgacttagaaggtcgttgctctatccaa180ctgagctaagggcgcgttgataccgcaatgcggtgtaatcgcgtgaattatacggtcaac240ccttgctgagtcaatggcttttagatctgaattctcatgtttgacagcttatcatcgata300agctttaatgcggtagtttatcacagttaaattgctaacgcagtcaggcaccgtgtatga360aatctaacaatgcgctcatcgtcatcctcggcaccgtcaccctggatgctgtaggcatag420gcttggttatgccggtactgccgggcctcttgcgggatatcgtccattccgacagcatcg480ccagtcactatggcgtgctgctagtccagtaatgacctcagaactccatctggatttgtt540cagaacgctcggttgccgccgggcgttttttattggtgagaatcgcagcaacttgtcgcg600ccaatcgagccatgtcgtcgtcaacgaccccccattcaagaacagcaagcagcattgaga660actttggaatccagtccctcttccacctgctgactagcgctatatgcgttgatgcaattt720ctatgcgcacccgttctcggagcactgtccgaccgctttggccgccgcccagtcctgctc780gcttcgctacttggagccactatcgactacgcgatcatggcgaccacacccgtcctgtgg840atctctcacctaccaaacaatgcccccctgcaaaaaataaattcatataaaaaacataca900gataaccatctgcggtgataaattatctctggcggtgttgacataaataccactggcggt960gatactgagcacatcagcaggacgcactgaccaccatgaaggtgacgctcttaaaaatta1020agccctgaagaagggcagcattcaaagcagaaggctttggggtgtgtgatacgaaacgaa1080gcattggccgtaagtgcgattccggattagctgccaatgtgccaatcgcggggggttttc1140gttcaggactacaactgccacacaccaccaaagctaactgacaggagaatccagatggat1200gcacaaacacgccgccgcgaacgtcgcgcagagaaacaggctcaatggaaagcagcaaat1260cccctgttggttggggtaagcgcaaaaccagttccgaaagatttttttaactataaacgc1320tgatggaagcgtttatgcggaagaggtaaagcccttcccgagtaacaaaaaaacaacagc1380ataaataaccccgctcttacacattccagccctgaaaaagggcatcaaattaaaccacac1440ctatggtgtatgcatttatttgcatacattcaatcaattgttatctaaggaaatacttac1500atatgatatctagaggatcccgggtaccgagctcgtcgaccgatgcccttgagagccttc1560aacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgact1620gtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggc1680gaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatc1740ttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcgagaag1800caggccattatcgccggcatggcggccgacgcgctgggctacgtcttgctggcgttcgcg1860acgcgaggctggatggccttccccattatgattcttctcgcttccggcggcatcgggatg1920cccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacagcttcaa1980ggatcgctcgcggctcttaccagcctaacttcgatcactggaccgctgatcgtcacggcg2040atttatgccgcctcggcgagcacatggaacgggttggcatggattgtaggcgccgcccta2100taccttgtctgcctccccgcgttgcgtcgcggtgcatggagccgggccacctcgacctga2160atggaagccggcggcacctcgctaacggattcaccactccaagaattggagccaatcaat2220tcttgcggagaactgtgaatgcgcaaaccaacccttggcagaacatatccatcgcgtccg2280ccatctccagcagccgcacgcggcgcatctcgggcagcgttgggtcctggccacgggtgc2340gcatgatcgtgctcctgtcgttgaggacccggctaggctggcggggttgccttactggtt2400agcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaacgtctg2460cgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctggaaacgc2520ggaagtcagcgccctgcaccattatgttccggatctgcatcgcaggatgctgctggctac2580cctgtggaacacctacatctgtattaacgaagcgctggcattgaccctgagtgatttttc2640tctggtcccgccgcatccataccgccagttgtttaccctcacaacgttccagtaaccggg2700catgttcatcatcagtaacccgtatcgtgagcatcctctctcgtttcatcggtatcatta2760cccccatgaacagaaattcccccttacacggaggcatcaagtgaccaaacaggaaaaaac2820cgcccttaacatggcccgctttatcagaagccagacattaacgcttctggagaaactcaa2880cgagctggacgcggatgaacaggcagacatctgtgaatcgcttcacgaccacgctgatga2940gctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgca3000gctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtca3060gggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcga3120tagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcac3180catatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgct3240cttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtat3300cagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaaga3360acatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgt3420ttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggt3480ggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgc3540gctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaa3600gcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgct3660ccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggta3720actatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactg3780gtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggc3840ctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagtta3900ccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtg3960gtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctt4020tgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttgg4080tcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagtttta4140aatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtg4200aggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcg4260tgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgc4320gagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccg4380agcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccggg4440aagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctgcag4500gcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgat4560caaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctc4620cgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgc4680ataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaa4740ccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacac4800gggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttctt4860cggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactc4920gtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaa4980caggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactca5040tactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggat5100acatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaa5160aagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggc5220gtatcacgaggccctttcgtcttcaagaa5249 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 5249 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 4 : ttcagatctaaaagccattgactcagcaagggttgaccgtataattcacgcgattacacc60gcattgcggtatcaacgcgcccttagctcagttggatagagcaacgaccttctaagtcgt120gggccgcaggttcgaatcctgcagggcgcgccattacaattcaatcagttacgccttctt180tatatcctccataatttcagagtgggacatatttgggacattatcaccaaaaatgtcgtc240tattttcctcgcatgctctgtcacatgtgaattctcatgtttgacagcttatcatcgata300agctttaatgcggtagtttatcacagttaaattgctaacgcagtcaggcaccgtgtatga360aatctaacaatgcgctcatcgtcatcctcggcaccgtcaccctggatgctgtaggcatag420gcttggttatgccggtactgccgggcctcttgcgggatatcgtccattccgacagcatcg480ccagtcactatggcgtgctgctagtccagtaatgacctcagaactccatctggatttgtt540cagaacgctcggttgccgccgggcgttttttattggtgagaatcgcagcaacttgtcgcg600ccaatcgagccatgtcgtcgtcaacgaccccccattcaagaacagcaagcagcattgaga660actttggaatccagtccctcttccacctgctgactagcgctatatgcgttgatgcaattt720ctatgcgcacccgttctcggagcactgtccgaccgctttggccgccgcccagtcctgctc780gcttcgctacttggagccactatcgactacgcgatcatggcgaccacacccgtcctgtgg840atctctcacctaccaaacaatgcccccctgcaaaaaataaattcatataaaaaacataca900gataaccatctgcggtgataaattatctctggcggtgttgacataaataccactggcggt960gatactgagcacatcagcaggacgcactgaccaccatgaaggtgacgctcttaaaaatta1020agccctgaagaagggcagcattcaaagcagaaggctttggggtgtgtgatacgaaacgaa1080gcattggccgtaagtgcgattccggattagctgccaatgtgccaatcgcggggggttttc1140gttcaggactacaactgccacacaccaccaaagctaactgacaggagaatccagatggat1200gcacaaacacgccgccgcgaacgtcgcgcagagaaacaggctcaatggaaagcagcaaat1260cccctgttggttggggtaagcgcaaaaccagttccgaaagatttttttaactataaacgc1320tgatggaagcgtttatgcggaagaggtaaagcccttcccgagtaacaaaaaaacaacagc1380ataaataaccccgctcttacacattccagccctgaaaaagggcatcaaattaaaccacac1440ctatggtgtatgcatttatttgcatacattcaatcaattgttatctaaggaaatacttac1500atatgatatctagaggatcccgggtaccgagctcgtcgaccgatgcccttgagagccttc1560aacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgact1620gtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggc1680gaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatc1740ttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcgagaag1800caggccattatcgccggcatggcggccgacgcgctgggctacgtcttgctggcgttcgcg1860acgcgaggctggatggccttccccattatgattcttctcgcttccggcggcatcgggatg1920cccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacagcttcaa1980ggatcgctcgcggctcttaccagcctaacttcgatcactggaccgctgatcgtcacggcg2040atttatgccgcctcggcgagcacatggaacgggttggcatggattgtaggcgccgcccta2100taccttgtctgcctccccgcgttgcgtcgcggtgcatggagccgggccacctcgacctga2160atggaagccggcggcacctcgctaacggattcaccactccaagaattggagccaatcaat2220tcttgcggagaactgtgaatgcgcaaaccaacccttggcagaacatatccatcgcgtccg2280ccatctccagcagccgcacgcggcgcatctcgggcagcgttgggtcctggccacgggtgc2340gcatgatcgtgctcctgtcgttgaggacccggctaggctggcggggttgccttactggtt2400agcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaacgtctg2460cgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctggaaacgc2520ggaagtcagcgccctgcaccattatgttccggatctgcatcgcaggatgctgctggctac2580cctgtggaacacctacatctgtattaacgaagcgctggcattgaccctgagtgatttttc2640tctggtcccgccgcatccataccgccagttgtttaccctcacaacgttccagtaaccggg2700catgttcatcatcagtaacccgtatcgtgagcatcctctctcgtttcatcggtatcatta2760cccccatgaacagaaattcccccttacacggaggcatcaagtgaccaaacaggaaaaaac2820cgcccttaacatggcccgctttatcagaagccagacattaacgcttctggagaaactcaa2880cgagctggacgcggatgaacaggcagacatctgtgaatcgcttcacgaccacgctgatga2940gctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgca3000gctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtca3060gggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcga3120tagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcac3180catatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgct3240cttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtat3300cagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaaga3360acatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgt3420ttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggt3480ggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgc3540gctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaa3600gcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgct3660ccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggta3720actatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactg3780gtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggc3840ctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagtta3900ccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtg3960gtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctt4020tgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttgg4080tcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagtttta4140aatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtg4200aggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcg4260tgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgc4320gagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccg4380agcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccggg4440aagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctgcag4500gcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgat4560caaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctc4620cgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgc4680ataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaa4740ccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacac4800gggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttctt4860cggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactc4920gtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaa4980caggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactca5040tactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggat5100acatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaa5160aagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggc5220gtatcacgaggccctttcgtcttcaagaa5249 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 36 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 5 : ggggtaccatatgattttagatgtggattacataac36 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 33 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 6 : tcccccgggctaggattttttaatgttaagcca33 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 7 : aaggatcctgacattatagttacttataatggagactcattcgccttccc50 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 24 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 8 : ggaattctttcccgagttcataag24 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 95 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 9 : gctatcctggacgttgactacatcaccgaagaaggtaagccggttatccg50tctgttcaaaaaagaaaacggtaaattcaaaatcgaacacgaccg95 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 95 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 10 : accggtgatttttttaacttcttcgattttagagtcgtcacgcagcagag50cgtagatgtacggacggaaggtacggtcgtgttcgattttgaatt95 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 37 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 11 : ggggtaccatatggctatcctggacgttgactacatc37 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 32 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 12 : ggggtaccaccggtgatttttttaacttcttc32 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 32 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 13 : aaataaccggtgaacgtcatggaaagattgtg32 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 21 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 14 : cctttgcttcattttcatctg21 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 15 : agtaggccacaagttctgcaaggacatccctggttttataccaagtctct50 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 16 : tgggacatttgctcgaggaaagacaaaagattaagacaaaaatgaaggaa50 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 17 : actcaagatcctatagaaaaaatactccttgactatagacaaaaagcgat50 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 18 : aaaactcttagcaaattctttctacggatattatggctatgcaaaagcgc50 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 19 : gctggtactgtaaggagtgtgctgagagcgttactgcctggggaagaaag50 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 26 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 20 : tacatcgagttagtatggaaggagct26 ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 21 : ccttccatactaactcgatgtactttcttccccaggcagtaacgctctca50 ( 2 ) information for seq id no : 22 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 22 : gcacactccttacagtaccagcgcgcttttgcatagccataatatccgta50 ( 2 ) information for seq id no : 23 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 23 : gaaagaatttgctaagagttttatcgctttttgtctatagtcaaggagta50 ( 2 ) information for seq id no : 24 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 24 : ttttttctataggatcttgagtttccttcatttttgtcttaatcttttgt50 ( 2 ) information for seq id no : 25 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 25 : ctttcctctaacaaatgtcccaagagacttggtataaaaccagggatgtc50 ( 2 ) information for seq id no : 26 :( i ) sequence characteristics :( a ) length : 21 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 26 : cttgcagaacttgtggcctac21 ( 2 ) information for seq id no : 27 :( i ) sequence characteristics :( a ) length : 40 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 27 : tcgctcctcaagtaggccacaagttctgcaaggacatccc40 ( 2 ) information for seq id no : 28 :( i ) sequence characteristics :( a ) length : 40 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 28 : tcttcgagctccttccatactaactcgatgtactttcttc40 ( 2 ) information for seq id no : 29 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 29 : aaaactcttatacaattctttctacggatattatggctatgcaaaagcgc50 ( 2 ) information for seq id no : 30 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 30 : gaaagaattgtataagagttttatcgctttttgtctatagtcaaggagta50 ( 2 ) information for seq id no : 31 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 31 : aaaactcttagcatactctttctacggatattatggctatgcaaaagcgc50 ( 2 ) information for seq id no : 32 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 32 : gaaagagtatgctaagagttttatcgctttttgtctatagtcaaggagta50 ( 2 ) information for seq id no : 33 :( i ) sequence characteristics :( a ) length : 53 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 33 : aaaactcttagcatacaattctttctacggatattatggctatgcaaaagcgc53 ( 2 ) information for seq id no : 34 :( i ) sequence characteristics :( a ) length : 53 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 34 : gaaagaattgtatgctaagagttttatcgctttttgtctatagtcaaggagta53 ( 2 ) information for seq id no : 35 :( i ) sequence characteristics :( a ) length : 53 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 35 : aaaaaccatcaactacggtgttctctacggatattatggctatgcaaaagcgc53 ( 2 ) information for seq id no : 36 :( i ) sequence characteristics :( a ) length : 53 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 36 : gagaacaccgtagttgatggtttttatcgctttttgtctatagtcaaggagta53 ( 2 ) information for seq id no : 37 :( i ) sequence characteristics :( a ) length : 776 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 37 : 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( 2 ) information for seq id no : 38 :( i ) sequence characteristics :( a ) length : 2328 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 38 : atgattttagatgtggattacataactgaagaaggaaaacctgttattaggctattcaaa60aaagagaacggaaaatttaagatagagcatgatagaacttttagaccatacatttacgct120cttctcagggatgattcaaagattgaagaagttaagaaaataacgggggaaaggcatgga180aagattgtgagaattgttgatgtagagaaggttgagaaaaagtttctcggcaagcctatt240accgtgtggaaactttatttggaacatccccaagatgttcccactattagagaaaaagtt300agagaacatccagcagttgtggacatcttcgaatacgatattccatttgcaaagagatac360ctcatcgacaaaggcctaataccaatggagggggaagaagagctaaagattcttgccttc420gatatagaaaccctctatcacgaaggagaagagtttggaaaaggcccaattataatgatt480agttatgcagatgaaaatgaagcaaaggtgattacttggaaaaacatagatcttccatac540gttgaggttgtatcaagcgagagagagatgataaagagatttctcaggattatcagggag600aaggatcctgacattatagttacttataatggagactcattcgacttcccatatttagcg660aaaagggcagaaaaacttgggattaaattaaccattggaagagatggaagcgagcccaag720atgcagagaataggcgatatgacggctgtagaagtcaagggaagaatacatttcgacttg780tatcatgtaataacaaggacaataaatctcccaacatacacactagaggctgtatatgaa840gcaatttttggaaagccaaaggagaaggtatacgccgacgagatagcaaaagcctgggaa900agtggagagaaccttgagagagttgccaaatactcgatggaagatgcaaaggcaacttat960gaactcgggaaagaattccttccaatggaaattcagctttcaagattagttggacaacct1020ttatgggatgtttcaaggtcaagcacagggaaccttgtagagtggttcttacttaggaaa1080gcctacgaaagaaacgaagtagctccaaacaagccaagtgaagaggagtatcaaagaagg1140ctcagggagagctacacaggtggattcgttaaagagccagaaaaggggttgtgggaaaac1200atagtatacctagattttagagccctatatccctcgattataattacccacaatgtttct1260cccgatactctaaatcttgagggatgcaagaactatgatatcgctcctcaagtaggccac1320aagttctgcaaggacatccctggttttataccaagtctcttgggacatttgttagaggaa1380agacaaaagattaagacaaaaatgaaggaaactcaagatcctatagaaaaaatactcctt1440gactatagacaaaaagcgataaaactcttagcaaattctttctacggatattatggctat1500gcaaaagcaagatggtactgtaaggagtgtgctgagagcgttactgcctggggaagaaag1560tacatcgagttagtatggaaggagctcgaagaaaagtttggatttaaagtcctctacatt1620gacactgatggtctctatgcaactatcccaggaggagaaagtgaggaaataaagaaaaag1680gctctagaatttgtaaaatacataaattcaaagctccctggactgctagagcttgaatat1740gaagggttttataagaggggattcttcgttacgaagaagaggtatgcagtaatagatgaa1800gaaggaaaagtcattactcgtggtttagagatagttaggagagattggagtgaaattgca1860aaagaaactcaagctagagttttggagacaatactaaaacacggagatgttgaagaagct1920gtgagaatagtaaaagaagtaatacaaaagcttgccaattatgaaattccaccagagaag1980ctcgcaatatatgagcagataacaagaccattacatgagtataaggcgataggtcctcac2040gtagctgttgcaaagaaactagctgctaaaggagttaaaataaagccaggaatggtaatt2100ggatacatagtacttagaggcgatggtccaattagcaatagggcaattctagctgaggaa2160tacgatcccaaaaagcacaagtatgacgcagaatattacattgagaaccaggttcttcca2220gcggtacttaggatattggagggatttggatacagaaaggaagacctcagataccaaaag2280acaagacaagtcggcctaacttcctggcttaacattaaaaaatcctag2328__________________________________________________________________________