Patent Application: US-64865796-A

Abstract:
an enzymatically active dna polymerase having between 540 and 582 amino acids having a tyrosine at a position equivalent to position 667 of taq dna polymerase , wherein said polymerase lacks 5 &# 39 ; to 3 &# 39 ; exonuclease activity , and wherein said polymerase has at least 95 % homology in its amino acid sequence to the dna polymerase of thermus aquaticus , thermus flavus or thermus thermophilus , and wherein said polymerase forms a single polypeptide band on an sds page .

Description:
fig1 - 4 are the dna sequences , and corresponding amino acid sequences , of fy2 , fy3 , and the dna polymerases of t . flavus and thermus thermophilus , respectively . fig5 is the dna sequence and corresponding amino acid sequence of fy4 . the following examples serve to illustrate the dna polymerases of the present invention and their use in sequencing . e . coli strains : mv1190 δ ( srl - reca ) 306 :: tn10 , δ ( lac - proab ), thi , supe , f &# 39 ; ( trad36 proab + laci q lacz δm15 )! ; dhλ + gyra96 , reca1 , rela1 , enda1 , thi - 1 , hsdr17 , supe44 , λ +! ; m 5248 λ ( bio275 , ci857 , ciii +, n +, δ ( h1 ))!. reaction conditions based on the procedure of barnes ( 91 proc . nat &# 39 ; l . acad . sci . 2216 - 2220 , 1994 ) were as follows : 20 mm tricine ph8 . 8 , 85 mm koac , 200 mm dntps , 10 % glycerol , 5 % dmso , 0 . 5 mm each primer , 1 . 5 mm mgoac , 2 . 5 u hottub ( amersham life science inc . ), 0 . 025 u deepvent ( new england biolabs ), 1 - 100 ng target dna per 100 ml reaction . cycling conditions were 94 ° c . 30 s , 68 ° c . 10 m40 s for 8 cycles ; then 94 ° c . 30 s , 68 ° c . 12 m00 s for 8 cycles ; then 94 ° c . 30 s , 68 ° c . 13 m20 s for 8 cycles ; then 94 ° c . 30 s , 68 ° c . 14 m40 s for 8 cycles . restriction enzyme digestions , plasmid preparations , and other in vitro manipulations of dna were performed using standard protocols ( sambrook et al ., molecular cloning 2nd ed . cold spring harbor press , 1989 ). pcr ( see protocol above ) was used to introduce a phe to tyr amino acid change at codon 667 of native taq dna polymerase ( which is codon 396 of fy2 ). oligonucleotide primer 1 dgcttgggcagaggatccgccggg ( seq . id . no . 3 ) spans nucleotides 954 to 976 of the coding region of seq . id . no . 1 including a bamhi restriction site . mutagenic oligo primer 2 dgggatggctagctcctgggagaggcggtgggccgacatgccgtaga ggaccccgtagttgatgg ( seq . id . no . 4 ) spans nucleotides 1178 to 1241 including an nhei site and codon 396 of sequence id . no . 1 . a clone of exo - taq deleted for the first 235 amino acids , pwb253 encoding deltataq polymerase ( barnes , 112 gene 29 - 35 , 1992 ) was used as template dna . any clone of taq polymerase or genomic dna from thermus aguaticus could also be utilized to amplify the identical pcr product . the pcr product was digested with bamhi and nhei , and this fragment was ligated to bamhi / nhei digested pwb253 plasmid to replace the corresponding fragment to create pwb253y , encoding polymerase fy1 . cells of e . coli strain mv1190 were used for transformation and induction of protein expression , although any host strain carrying a lac repressor could be substituted . dna sequencing verified the phe to tyr change in the coding region . pcr primer 3 dggaattccatatggacgatctgaagctctcc ( seq . id . no . 5 ) spanning the start codon and containing restriction enzyme sites , was used with pcr primer 4 dggggtaccaagcttcactccttggcggagag ( seq . id . no . 6 ) containing restriction sites and spanning the stop codon ( codon 562 of sequence id . no . 1 ). a methionine start codon and restriction enzyme recognition sequences were added to pcr primer 5 dggaattccatatgctggagaggcttgagttt ( seq . id . no . 7 ), which was used with primer 4 above . pcr was performed using the above primer pairs , and plasmid pwb253y as template . the pcr products were digested with restriction enzymes ndei and kpni and ligated to ndei / kpni digested vector pre2 ( reddi et al ., 17 nucleic acids research 10 , 473 - 10 , 488 , 1989 ) to make plasmids pre236y , encoding fy1 polymerase , and pre273y encoding fy2 polymerase , respectively . cells of e . coli strain dhλ + were used for primary transformation with this and all subsequent pre2 constructions , and strain m5248 ( λci857 ) was used for protein expression , although any comparable pair of e . coli strains carrying the ci + and ci857 alleles could be utilized . alternatively , any rec + ci + strain could be induced by chemical agents such as nalidixic acid to produce the polymerase . the sequences of both genes were verified . pre273y was found to produce a single polypeptide band on sds polyacrylamide gels , unlike pre253y or pre236y . primer 6 dggaattccatatgctggaacgtctggagtttggcagcctc ctc ( seq . id . no . 8 ) and primer 4 were used to make a pcr product introducing silent changes in codon usage of fy2 . the product was digested with ndei / bamhi and ligated to a pre2 construct containing the 3 &# 39 ; end of fy2 to create prefy2pref , encoding fy2 dna polymerase . primer 7 dggaattccatatggctctggaacgtctggagtttggcagcctcctc ( seq . id . no . 9 ) and primer 4 were used to make a pcr product introducing an additional alanine codon commonly occurring at the second position of highly expressed genes . the ndei / bamhi digested fragment was used as above to create prefy3 , encoding fy3 dna polymerase . e . coli strains : dh1λ + gyra96 , reca1 , rela1 , enda1 , thi - 1 , hsdr17 , supe44 , λ + ! ; m5248 λ ( bio275 , ci857 , ciii +, n +, δ ( h1 ))!. genomic dna was prepared by standard techniques from thermus thermophilus . the dna polymerase gene of thermus thermophilus is known to reside on a 3 kilobase alwni fragment . to enrich for polymerase sequences in some pcr reactions , the genomic dna was digested prior to pcr with alwni , and fragments of approximately 3 kb were selected by agarose gel electrophoresis to be used as template dna . reaction conditions were as follows : 10 mm tris ph8 . 3 , 50 mm kcl , 800 μm dntps , 0 . 001 % gelatin , 1 . 0 μm each primer , 1 . 5 mm mgcl 2 , 2 . 5 u tth , 0 . 025 u deepvent ( new england biolabs ), per 100 μl reaction . cycling conditions were 94 ° c . 2 min , then 35 cycles of 94 ° c . 30 s , 55 ° c . 30 s , 72 ° c . 3 min , followed by 72 ° c . for 7 min . restriction enzyme digestions , plasmid preparations , and other in vitro manipulations of dna were performed using standard protocols ( sambrook et al ., 1989 ). plasmid pmr1 was constructed to encode an exonuclease - free polymerase , with optimized codons for expression in e . coli at the 5 &# 39 ; end . primer 8 ( seq . id . no . 10 ) ( ggaattccatatgctggaacgtctggaattcggcagcctc ) was used with primer 9 ( seq . id . no . 11 ) ( ggggtaccctaacccttggcggaaagccagtc ) to create a pcr product from tth genomic dna , which was digested with restriction enzymes ndei and kpni and inserted into plasmid pre2 ( reddi et al ., 1989 , nucleic acids research 17 , 10473 - 10488 ) digested with the same enzymes . to create the desired f396y mutation , two pcr products were made from tth chromosomal dna . primer 8 above was used in combination with primer 10 ( seq . id . no . 12 ) ( gggatggctagctcctgggagagcctatgggcggacat gccgtagaggacgccgtagttcaccg ) to create a portion of the gene containing the f to y amino acid change as well as a silent change to create an nhei restriction site . primer 11 ( seq . id . no . 13 )( ctagctagccatccccta cgaagaagcggtggcct ) was used in combination with primer 9 above to create a portion of the gene from the introduced nhei site to the stop codon at the 3 &# 39 ; end of the coding sequence . the pcr product of primers 8 and 10 was digested with ndei and nhei , and the pcr product of primers 9 and 11 was digested with nhei and kpni . these were introduced into expression vector pre2 which w as digested with ndei and kpni to produce plasmid pmr5 . in addition to the desired changes , pmr5 was found to have a spurious change introduced by pcr , which led to an amino acid substitution , k234r . plasmid pmr8 was created to eliminate this substitution , by replacing the aflii / bamhi fragment of pmr5 for the corresponding fragment from pmr1 . the fy4 polymerase encoded by plasmid pmr8 ( seq . id . no . 14 ) is given in fig5 . cells of e . coli strain dh1λ + were used for primary transformation , and strain m5248 ( λci857 ) was used for protein expression , although any comparable pair of e . coli strains carrying the ci + and ci857 alleles could be utilized . alternatively , any rec + ci + strain could be induced by chemical agents such as nalidixic acid to produce the polymerase . determinations of amino terminal protein sequences were performed at the w . m . keck foundation , biotechnology resource laboratory , new haven , conn . a 1 liter culture of 2 × lb ( 2 % bacto - tryptone , 1 % bacto - yeast extract , 0 . 5 % nacl )+ 0 . 2 % casamino acids + 20 mm kpo 4 ph 7 . 5 + 50 μg / ml ampicillin was inoculated with a glycerol stock of the appropriate cell strain and grown at 30 ° c . with agitation until cells were in log phase ( 0 . 7 - 1 . 0 od 590 ). 9 liters of 2 × lb + 0 . 2 % casamino acids + 20 mm kpo 4 ph 7 . 5 + 0 . 05 % mazu anti - foam was inoculated with 1 liter of log phase cells in 10 liter microferm fermentors ( new brunswick scientific co .). cells were grown at 30 ° c . under 15 psi pressure , 350 - 450 rpm agitation , and an air flow rate of 14 , 000 cc / min ± 1000 cc / min . when the od 590 reached 1 . 5 - 2 . 0 , the cultures were induced by increasing the temperature to 40 °- 42 ° c . for 90 - 120 minutes . the cultures were then cooled to & lt ; 20 ° c . and the cells harvested by centrifugation in a sorvall rc - 3b centrifuge at 5000 rpm at 4 ° c . for 15 - 20 minutes . harvested cells were stored at - 80 ° c . frozen cells were broken into small pieces and resuspended in pre - warmed ( 90 °- 95 ° c .) lysis buffer ( 20 mm tris ph 8 . 5 , 1 mm edta , 10 mm mgcl 2 , 16 mm ( nh 4 ) 2 so 4 , 0 . 1 % tween 20 , 0 . 1 % nonidet p - 40 , 1 mm pmsf ). resuspended cells were then heated rapidly to 80 ° c . and incubated at 80 ° c . for 20 minutes with constant stirring . the suspension was then rapidly cooled on ice . the cell debris was removed by centrifugation using a sorvall gsa rotor at 10 , 000 rpm for 20 minutes at 4 ° c . the nacl concentration of the supernatant was adjusted to 300 mm . the sample was then passed through a diethylaminoethyl cellulose ( whatman de - 52 ) column that had been previously equilibrated with buffer a ( 20 mm tris ph 8 . 5 , 1 mm edta , 0 . 1 % tween 20 , 0 . 1 % nonidet p - 40 , 300 mm nacl , 10 % glycerol , 1 mm dtt ), and polymerase collected in the flow through . the sample was then diluted to a concentration of nacl of 100 mm and applied to a heparin - sepharose column . the polymerase was eluted from the column with a nacl gradient ( 100 - 500 mm nacl ). the sample was then dialyzed against buffer b ( 20 mm tris ph 8 . 5 , 1 mm edta , 0 . 1 % tween 20 , 0 . 1 % nonidet p - 40 , 10 mm kcl , 10 % glycerol , 1 mm dtt ) and further diluted as needed to lower the conductivity of the sample to the conductivity of buffer b . the sample was then applied to a diethylaminoethyl ( waters deae 15 hr ) column and eluted with a 10 - 500 mm kcl gradient . the polymerase was then diluted with an equal volume of final buffer ( 20 mm tris ph 8 . 5 , 0 . 1 mm edta , 0 . 5 % tween 20 , 0 . 5 % nonidet p - 40 , 100 mm kcl , 50 % glycerol , 1 mm dtt ) and dialyzed against final buffer . the exonuclease assay was performed by incubating 5 ul ( 25 - 150 units ) of dna polymerase with 5 ug of labelled 3 h !- pbr322 pcr fragment ( 1 . 6 × 10 4 cpm / ug dna ) in 100 ul of reaction buffer of 20 mm tris . hcl ph 8 . 5 , 5 mm mgcl 2 , 10 mm kcl , for 1 hour at 60 ° c . after this time interval , 200 ul of 1 : 1 ratio of 50 ug / ml salmon sperm dna with 2 mm edta and 20 % tca with 2 % sodium pyrophosphate were added into the assay aliquots . the aliquots were put on ice for 10 min and then centrifuged at 12 , 000 g for 10 min . acid - soluble radioactivity in 200 ul of the supernatant was quantitated by liquid scintillation counting . one unit of exonuclease activity was defined as the amount of enzyme that catalyzed the acid solubilization of 10 nmol of total nucleotide in 30 min at 60 ° c . the following components were added to a microcentrifuge vial ( 0 . 5 ml ) : 0 . 4 pmol m13 dna ( e . g ., m13 mp18 , 1 . 0 μg ); 2 μl reaction buffer ( 260 mm tris - hcl , ph 9 . 5 65 mm mgcl 2 ); 2 μl of labeling nucleotide mixture ( 1 . 5 μm each of dgtp , dctp and dttp ); 0 . 5 μl ( 5 μci ) of a - 33 p ! datp ( about 2000 ci / mmol ); 1 μl - 40 primer ( 0 . 5 μm ; 0 . 5 pmol / μl 5 &# 39 ; gttttcccagtcacgac - 3 &# 39 ;); 2 μl of a mixture containing 4 u / μl fy polymerase and 6 . 6 u / ml thermoplasma acidophilum inorganic pyrophophatase ( 32 u / μl polymerase and 53 u / ml pyrophosphatase in 20 mm tris ( ph8 . 5 ), 100 mm kcl , 0 . 1 mm edta , 1 mm dtt , 0 . 5 % np - 40 , 0 . 5 % tween - 20 and 50 % glycerol , diluted 8 fold in dilution buffer ( 10 mm tris - hcl ph8 . 0 , 1 mm 2 - mercaptoethanol , 0 . 5 % tween - 20 , 0 . 5 % np - 40 )); and water to a total volume of 17 . 5 μl . these components ( the labeling reaction ) were mixed and the vial was placed in a constant - temperature water bath at 45 ° c . for 5 minutes . four vials were labeled a , c , g , and t , and filled with 4 μl of the corresponding termination mix : dda termination mix ( 150 μm each datp , dctp , dgtp , dttp , 1 . 5 μm ddatp ); ddt termination mix ( 150 μm each datp , dctp , dgtp , dttp , 1 . 5 μm ddttp ); ddc termination mix ( 150 μm each datp , dctp , dgtp , dttp , 1 . 5 μm ddctp ); ddg termination mix ( 150 μm each datp , dctp , dgtp , dttp , 1 . 5 μm ddgtp ). the labeling reaction was divided equally among the four termination vials ( 4 μl to each termination reaction vial ), and tightly capped . the four vials were placed in a constant - temperature water bath at 72 ° c . for 5 minutes . then 4 μl of stop solution ( 95 % formamide 20 mm edta , 0 . 05 % bromophenol blue , 0 . 05 % xylene cyanol ff ) added to each vial , and heated briefly to 70 °- 80 ° c . immediately prior to loading on a sequencing gel ( 8 % acrylamide , 8 . 3m urea ). autoradiograms required an 18 - 36 hour exposure using kodak xar - 5 film or amersham hyperfilm mp . high - quality sequence results with uniform band intensities were obtained . the band intensities were much more uniform than those obtained with similar protocols using taq dna polymerase or δtaq dna polymerase . the following components were added to a microcentrifuge vial ( 0 . 5 ml ) which is suitable for insertion into a thermocycler machine ( e . g ., perkin - elmer dna thermal cycler ): 0 . 05 pmol or more m13 dna ( e . g ., m13 mp18 , 0 . 1 μg ), or 0 . 1 μg double - stranded plasmid dna ( e . g ., puc19 ); 2 μl reaction buffer ( 260 mm tris - hcl , ph 9 . 5 65 mm mgcl 2 ); 1 μl 3 . 0 μm dgtp ; 1 μl 3 . 0 μm dttp ; 0 . 5 μl ( 5 μci ) of α - 33 p ! datp ( about 2000 ci / mmol ); 1 μl - 40 primer ( 0 . 5 μm ; 0 . 5 pmol / μl 5 &# 39 ; gttttcccagtcacgac - 3 &# 39 ;); 2 μl of a mixture containing 4 u / μl fy polymerase and 6 . 6 u / ml thermoplasma acidophilum inorganic pyrophophatase ( 32 u / μl polymerase and 53 u / ml pyrophosphatase in 20 mm tris ( ph8 . 5 ), 100 mm kcl , 0 . 1 mm edta , 1 mm dtt , 0 . 5 % np - 40 , 0 . 5 % tween - 20 and 50 % glycerol , diluted 8 fold in dilution buffer ( 10 mm tris - hcl ph8 . 0 , 1 mm 2 - mercaptoethanol , 0 . 5 % tween - 20 , 0 . 5 % np - 40 )); and water to a total volume of 17 . 5 μl . these components ( labeling reaction mixture ) were mixed and overlaid with 10 μl light mineral oil ( amersham ). the vial was placed in the thermocycler and 30 - 100 cycles ( more than 60 cycles is unnecessary ) from 45 ° c . for 1 minute to 95 ° c . for 0 . 5 minute performed . ( temperatures can be cycled from 55 °- 95 ° c ., if desired ) the temperatures may be adjusted if the melting temperature of the primer / template is significantly higher or lower , but these temperatures work well for most primer - templates combinations . this step can be completed in about 3 minutes per cycle . four vials were labeled a , c , g , and t , and filled with 4 ml of the corresponding termination mix : dda termination mix ( 150 μm each datp , dctp , dgtp , dttp , 1 . 5 μm ddatp ); ddt termination mix ( 150 μm each datp , dctp , dgtp , dttp , 1 . 5 μm ddttp ); ddc termination mix ( 150 μm each datp , dctp , dgtp , dttp , 1 . 5 μm ddctp ); ddg termination mix ( 150 μm each datp , dctp , dgtp , dttp , 1 . 5 μm ddgtp ). no additional enzyme is added to the termination vials . the enzyme carried in from the prior ( labeling ) step is sufficient . the cycled labeling reaction mixture was divided equally among the four termination vials ( 4 μl to each termination reaction vial ), and overlaid with 10 μl of light mineral oil . the four vials were placed in the thermocycler and 30 - 200 cycles ( more than 60 cycles is unnecessary ) performed from 95 ° c . for 15 seconds , 55 ° c . for 30 seconds , and 72 ° c . for 120 seconds . this step was conveniently completed overnight . other times and temperatures are also effective . six μl of reaction mixture was removed ( avoiding oil ), 3 μl of stop solution ( 95 % formamide 20 mm edta , 0 . 05 % bromophenol blue , 0 . 05 % xylene cyanol ff ) added , and heated briefly to 70 °- 80 ° c . immediately prior to loading on a sequencing gel . autoradiograms required an 18 - 36 hour exposure using kodak xar - 5 film or amersham hyperfilm mp . high - quality sequence results with uniform band intensities were obtained . the band intensities were much more uniform than those obtained with similar protocols using taq dna polymerase or δtaq dna polymerase . for either of the sequencing methods outlined in examples 1 and 2 , 7 - deaza - 2 &# 39 ; deoxy - gtp can be substituted for dgtp in the labeling and termination mixtures at exactly the same concentration as dgtp . when this substitution is made , secondary structures on the gels are greatly reduced . similarly , 2 &# 39 ;- deoxyinosinetriphosphate can also be substituted for dgtp but its concentration must be 10 - fold higher than the corresponding concentration of dgtp . substitution of ditp for dgtp is even more effective in eliminating compression artifacts than 7 - deaza - dgtp . fy polymerases have been adapted for use with many other sequencing methods , including the use of fluorescent primers and fluorescent - dideoxy - terminators for sequencing with the abi 373a dna sequencing instrument . protein samples were run on a 14 × 16 mm 7 . 5 or 10 % polyacrylamide gel . ( gels were predominantly 10 % polyacrylamide using a 14 × 16 mm hoefer apparatus . other sizes , apparatuses , and percentage gels are acceptable . similar results can also be obtained using the pharmacia phast gel system with sds , 8 - 25 % gradient gels . reagent grade and ultrapure grade reagents were used .) the stacking gel consisted of 4 % acrylamide ( 30 : 0 . 8 , acrylamide : bisacrylamide ), 125 mm tris - hcl ph 6 . 8 , 0 . 1 % sodium dodecyl sulfate ( sds ). the resolving gel consisted of 7 . 5 or 10 % acrylamide ( 30 : 0 . 8 , acrylamide : bisacrylamide ), 375 mm tris - hcl ph 8 . 8 , 0 . 1 % sds . running buffer consisted of 25 mm tris , 192 mm glycine and 0 . 1 % sds . 1 × sample buffer consisted of 25 mm tris - hcl ph 6 . 8 , 0 . 25 % sds , 10 % glycerol , 0 . 1m dithiothreitol , 0 . 1 % bromophenol blue , and 1 mm edta . a 1 / 4 volume of 5 × sample buffer was added to each sample . samples were heated in sample buffer to 90 °- 100 ° c . for approximately 5 minutes prior to loading . a 1 . 5 mm thick gel was run at 50 - 100 ma constant current for 1 - 3 hours ( until bromophenol blue was close to the bottom of the gel ). the gel was stained with 0 . 025 % coomassie blue r250 in 50 % methanol , 10 % acetic acid and destained in 5 % methanol , 7 % acetic acid solution . a record of the gel was made by taking a photograph of the gel , by drying the gel between cellulose film sheets , or by drying the gel onto filter paper under a vacuum . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 14 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 1686 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ix ) feature :( a ) name / key : fy2 ( b ) location : 1 ... 1683 ( xi ) sequence description : seq id no : 1 : atgctggagaggcttgagtttggcagcctcctccacgagttcggcctt48metleugluargleuglupheglyserleuleuhisglupheglyleu151015ctggaaagccccaaggccctggaggaggccccctggcccccgccggaa96leugluserprolysalaleugluglualaprotrpproproproglu202530ggggccttcgtgggctttgtgctttcccgcaaggagcccatgtgggcc144glyalaphevalglyphevalleuserarglysglupromettrpala354045gatcttctggccctggccgccgccagggggggccgggtccaccgggcc192aspleuleualaleualaalaalaargglyglyargvalhisargala505560cccgagccttataaagccctcagggacctgaaggaggcgcgggggctt240progluprotyrlysalaleuargaspleulysglualaargglyleu65707580ctcgccaaagacctgagcgttctggccctgagggaaggccttggcctc288leualalysaspleuservalleualaleuarggluglyleuglyleu859095ccgcccggcgacgaccccatgctcctcgcctacctcctggacccttcc336proproglyaspaspprometleuleualatyrleuleuaspproser100105110aacaccacccccgagggggtggcccggcgctacggcggggagtggacg384asnthrthrprogluglyvalalaargargtyrglyglyglutrpthr115120125gaggaggcgggggagcgggccgccctttccgagaggctcttcgccaac432gluglualaglygluargalaalaleusergluargleuphealaasn130135140ctgtgggggaggcttgagggggaggagaggctcctttggctttaccgg480leutrpglyargleugluglyglugluargleuleutrpleutyrarg145150155160gaggtggagaggcccctttccgctgtcctggcccacatggaggccacg528gluvalgluargproleuseralavalleualahismetglualathr165170175ggggtgcgcctggacgtggcctatctcagggccttgtccctggaggtg576glyvalargleuaspvalalatyrleuargalaleuserleugluval180185190gccgaggagatcgcccgcctcgaggccgaggtcttccgcctggccggc624alaglugluilealaargleuglualagluvalpheargleualagly195200205caccccttcaacctcaactcccgggaccagctggaaagggtcctcttt672hispropheasnleuasnserargaspglnleugluargvalleuphe210215220gacgagctagggcttcccgccatcggcaagacggagaagaccggcaag720aspgluleuglyleuproalaileglylysthrglulysthrglylys225230235240cgctccaccagcgccgccgtcctggaggccctccgcgaggcccacccc768argserthrseralaalavalleuglualaleuargglualahispro245250255atcgtggagaagatcctgcagtaccgggagctcaccaagctgaagagc816ilevalglulysileleuglntyrarggluleuthrlysleulysser260265270acctacattgaccccttgccggacctcatccaccccaggacgggccgc864thrtyrileaspproleuproaspleuilehisproargthrglyarg275280285ctccacacccgcttcaaccagacggccacggccacgggcaggctaagt912leuhisthrargpheasnglnthralathralathrglyargleuser290295300agctccgatcccaacctccagaacatccccgtccgcaccccgcttggg960serseraspproasnleuglnasnileprovalargthrproleugly305310315320cagaggatccgccgggccttcatcgccgaggaggggtggctattggtg1008glnargileargargalapheilealaglugluglytrpleuleuval325330335gccctggactatagccagatagagctcagggtgctggcccacctctcc1056alaleuasptyrserglnilegluleuargvalleualahisleuser340345350ggcgacgagaacctgatccgggtcttccaggaggggcgggacatccac1104glyaspgluasnleuileargvalpheglngluglyargaspilehis355360365acggagaccgccagctggatgttcggcgtcccccgggaggccgtggac1152thrgluthralasertrpmetpheglyvalproargglualavalasp370375380cccctgatgcgccgggcggccaagaccatcaactacggggtcctctac1200proleumetargargalaalalysthrileasntyrglyvalleutyr385390395400ggcatgtcggcccaccgcctctcccaggagctagccatcccttacgag1248glymetseralahisargleuserglngluleualaileprotyrglu405410415gaggcccaggccttcattgagcgctactttcagagcttccccaaggtg1296glualaglnalapheilegluargtyrpheglnserpheprolysval420425430cgggcctggattgagaagaccctggaggagggcaggaggcgggggtac1344argalatrpileglulysthrleuglugluglyargargargglytyr435440445gtggagaccctcttcggccgccgccgctacgtgccagacctagaggcc1392valgluthrleupheglyargargargtyrvalproaspleugluala450455460cgggtgaagagcgtgcgggaggcggccgagcgcatggccttcaacatg1440argvallysservalargglualaalagluargmetalapheasnmet465470475480cccgtccagggcaccgccgccgacctcatgaagctggctatggtgaag1488provalglnglythralaalaaspleumetlysleualametvallys485490495ctcttccccaggctggaggaaatgggggccaggatgctccttcaggtc1536leupheproargleugluglumetglyalaargmetleuleuglnval500505510cacgacgagctggtcctcgaggccccaaaagagagggcggaggccgtg1584hisaspgluleuvalleuglualaprolysgluargalaglualaval515520525gcccggctggccaaggaggtcatggagggggtgtatcccctggccgtg1632alaargleualalysgluvalmetgluglyvaltyrproleualaval530535540cccctggaggtggaggtggggataggggaggactggctctccgccaag1680proleugluvalgluvalglyileglygluasptrpleuseralalys545550555560gagtga1686glu *( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 1689 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ix ) feature :( a ) name / key : fy3 ( b ) location : 1 ... 1686 ( xi ) sequence description : seq id no : 2 : atggctctggaacgtctggagtttggcagcctcctccacgagttcggc48metalaleugluargleuglupheglyserleuleuhisgluphegly151015cttctggaaagccccaaggccctggaggaggccccctggcccccgccg96leuleugluserprolysalaleugluglualaprotrppropropro202530gaaggggccttcgtgggctttgtgctttcccgcaaggagcccatgtgg144gluglyalaphevalglyphevalleuserarglysglupromettrp354045gccgatcttctggccctggccgccgccagggggggccgggtccaccgg192alaaspleuleualaleualaalaalaargglyglyargvalhisarg505560gcccccgagccttataaagccctcagggacctgaaggaggcgcggggg240alaprogluprotyrlysalaleuargaspleulysglualaarggly65707580cttctcgccaaagacctgagcgttctggccctgagggaaggccttggc288leuleualalysaspleuservalleualaleuarggluglyleugly859095ctcccgcccggcgacgaccccatgctcctcgcctacctcctggaccct336leuproproglyaspaspprometleuleualatyrleuleuasppro100105110tccaacaccacccccgagggggtggcccggcgctacggcggggagtgg384serasnthrthrprogluglyvalalaargargtyrglyglyglutrp115120125acggaggaggcgggggagcgggccgccctttccgagaggctcttcgcc432thrgluglualaglygluargalaalaleusergluargleupheala130135140aacctgtgggggaggcttgagggggaggagaggctcctttggctttac480asnleutrpglyargleugluglyglugluargleuleutrpleutyr145150155160cgggaggtggagaggcccctttccgctgtcctggcccacatggaggcc528arggluvalgluargproleuseralavalleualahismetgluala165170175acgggggtgcgcctggacgtggcctatctcagggccttgtccctggag576thrglyvalargleuaspvalalatyrleuargalaleuserleuglu180185190gtggccgaggagatcgcccgcctcgaggccgaggtcttccgcctggcc624valalaglugluilealaargleuglualagluvalpheargleuala195200205ggccaccccttcaacctcaactcccgggaccagctggaaagggtcctc672glyhispropheasnleuasnserargaspglnleugluargvalleu210215220tttgacgagctagggcttcccgccatcggcaagacggagaagaccggc720pheaspgluleuglyleuproalaileglylysthrglulysthrgly225230235240aagcgctccaccagcgccgccgtcctggaggccctccgcgaggcccac768lysargserthrseralaalavalleuglualaleuargglualahis245250255cccatcgtggagaagatcctgcagtaccgggagctcaccaagctgaag816proilevalglulysileleuglntyrarggluleuthrlysleulys260265270agcacctacattgaccccttgccggacctcatccaccccaggacgggc864serthrtyrileaspproleuproaspleuilehisproargthrgly275280285cgcctccacacccgcttcaaccagacggccacggccacgggcaggcta912argleuhisthrargpheasnglnthralathralathrglyargleu290295300agtagctccgatcccaacctccagaacatccccgtccgcaccccgctt960serserseraspproasnleuglnasnileprovalargthrproleu305310315320gggcagaggatccgccgggccttcatcgccgaggaggggtggctattg1008glyglnargileargargalapheilealaglugluglytrpleuleu325330335gtggccctggactatagccagatagagctcagggtgctggcccacctc1056valalaleuasptyrserglnilegluleuargvalleualahisleu340345350tccggcgacgagaacctgatccgggtcttccaggaggggcgggacatc1104serglyaspgluasnleuileargvalpheglngluglyargaspile355360365cacacggagaccgccagctggatgttcggcgtcccccgggaggccgtg1152histhrgluthralasertrpmetpheglyvalproargglualaval370375380gaccccctgatgcgccgggcggccaagaccatcaactacggggtcctc1200aspproleumetargargalaalalysthrileasntyrglyvalleu385390395400tacggcatgtcggcccaccgcctctcccaggagctagccatcccttac1248tyrglymetseralahisargleuserglngluleualaileprotyr405410415gaggaggcccaggccttcattgagcgctactttcagagcttccccaag1296gluglualaglnalapheilegluargtyrpheglnserpheprolys420425430gtgcgggcctggattgagaagaccctggaggagggcaggaggcggggg1344valargalatrpileglulysthrleuglugluglyargargarggly435440445tacgtggagaccctcttcggccgccgccgctacgtgccagacctagag1392tyrvalgluthrleupheglyargargargtyrvalproaspleuglu450455460gcccgggtgaagagcgtgcgggaggcggccgagcgcatggccttcaac1440alaargvallysservalargglualaalagluargmetalapheasn465470475480atgcccgtccagggcaccgccgccgacctcatgaagctggctatggtg1488metprovalglnglythralaalaaspleumetlysleualametval485490495aagctcttccccaggctggaggaaatgggggccaggatgctccttcag1536lysleupheproargleugluglumetglyalaargmetleuleugln500505510gtccacgacgagctggtcctcgaggccccaaaagagagggcggaggcc1584valhisaspgluleuvalleuglualaprolysgluargalagluala515520525gtggcccggctggccaaggaggtcatggagggggtgtatcccctggcc1632valalaargleualalysgluvalmetgluglyvaltyrproleuala530535540gtgcccctggaggtggaggtggggataggggaggactggctctccgcc1680valproleugluvalgluvalglyileglygluasptrpleuserala545550555560aaggagtga1689lysglu *( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 23 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 3 : gcttgggcagaggatccgccggg23 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 64 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 4 : gggatggctagctcctgggagaggcggtgggccgacatgccgtagaggac50cccgtagttgatgg64 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 31 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 5 : ggaattccatatggacgatctgaagctctcc31 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 31 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 6 : ggggtaccaagcttcactccttggcggagag31 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 31 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 7 : ggaattccatatgctggagaggcttgagttt31 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 43 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 8 : ggaattccatatgctggaacgtctggagtttggcagcctcctc43 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 46 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 9 : ggaattccatatggctctggaacgtctggagtttggcagcctcctc46 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 40 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 10 : ggaattccatatgctggaacgtctggaattcggcagcctc40 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 32 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 11 : ggggtaccctaacccttggcggaaagccagtc32 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 64 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 12 : gggatggctagctcctgggagagcctatgggcggacatgccgtagaggac50gccgtagttcaccg64 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 35 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 13 : ctagctagccatcccctacgaagaagcggtggcct35 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 1686 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ix ) feature :( a ) name / key : fy4 ( b ) location : 1 ... 1683 ( xi ) sequence description : seq id no : 14 : atgctggaacgtctggaattcggcagcctcctccacgagttcggcctc48metleugluargleuglupheglyserleuleuhisglupheglyleu151015ctggaggcccccgcccccctggaggaggccccctggcccccgccggaa96leuglualaproalaproleugluglualaprotrpproproproglu202530ggggccttcgtgggcttcgtcctctcccgccccgagcccatgtgggcg144glyalaphevalglyphevalleuserargproglupromettrpala354045gagcttaaagccctggccgcctgcagggacggccgggtgcaccgggca192gluleulysalaleualaalacysargaspglyargvalhisargala505560gcagaccccttggcggggctaaaggacctcaaggaggtccggggcctc240alaaspproleualaglyleulysaspleulysgluvalargglyleu65707580ctcgccaaggacctcgccgtcttggcctcgagggaggggctagacctc288leualalysaspleualavalleualaserarggluglyleuaspleu859095gtgcccggggacgaccccatgctcctcgcctacctcctggacccctcc336valproglyaspaspprometleuleualatyrleuleuaspproser100105110aacaccacccccgagggggtggcgcggcgctacgggggggagtggacg384asnthrthrprogluglyvalalaargargtyrglyglyglutrpthr115120125gaggacgccgcccaccgggccctcctctcggagaggctccatcggaac432gluaspalaalahisargalaleuleusergluargleuhisargasn130135140ctccttaagcgcctcgagggggaggagaagctcctttggctctaccac480leuleulysargleugluglygluglulysleuleutrpleutyrhis145150155160gaggtggaaaagcccctctcccgggtcctggcccacatggaggccacc528gluvalglulysproleuserargvalleualahismetglualathr165170175ggggtacggctggacgtggcctaccttcaggccctttccctggagctt576glyvalargleuaspvalalatyrleuglnalaleuserleugluleu180185190gcggaggagatccgccgcctcgaggaggaggtcttccgcttggcgggc624alaglugluileargargleugluglugluvalpheargleualagly195200205caccccttcaacctcaactcccgggaccagctggaaagggtgctcttt672hispropheasnleuasnserargaspglnleugluargvalleuphe210215220gacgagcttaggcttcccgccttggggaagacgcaaaagacaggcaag720aspgluleuargleuproalaleuglylysthrglnlysthrglylys225230235240cgctccaccagcgccgcggtgctggaggccctacgggaggcccacccc768argserthrseralaalavalleuglualaleuargglualahispro245250255atcgtggagaagatcctccagcaccgggagctcaccaagctcaagaac816ilevalglulysileleuglnhisarggluleuthrlysleulysasn260265270acctacgtggaccccctcccaagcctcgtccacccgaggacgggccgc864thrtyrvalaspproleuproserleuvalhisproargthrglyarg275280285ctccacacccgcttcaaccagacggccacggccacggggaggcttagt912leuhisthrargpheasnglnthralathralathrglyargleuser290295300agctccgaccccaacctgcagaacatccccgtccgcacccccttgggc960serseraspproasnleuglnasnileprovalargthrproleugly305310315320cagaggatccgccgggccttcgtggccgaggcgggttgggcgttggtg1008glnargileargargalaphevalalaglualaglytrpalaleuval325330335gccctggactatagccagatagagctccgcgtcctcgcccacctctcc1056alaleuasptyrserglnilegluleuargvalleualahisleuser340345350ggggacgaaaacctgatcagggtcttccaggaggggaaggacatccac1104glyaspgluasnleuileargvalpheglngluglylysaspilehis355360365acccagaccgcaagctggatgttcggcgtccccccggaggccgtggac1152thrglnthralasertrpmetpheglyvalproproglualavalasp370375380cccctgatgcgccgggcggccaagacggtgaactacggcgtcctctac1200proleumetargargalaalalysthrvalasntyrglyvalleutyr385390395400ggcatgtccgcccataggctctcccaggagctagccatcccctacgaa1248glymetseralahisargleuserglngluleualaileprotyrglu405410415gaagcggtggcctttatagagcgctacttccaaagcttccccaaggtg1296glualavalalapheilegluargtyrpheglnserpheprolysval420425430cgggcctggatagaaaagaccctggaggaggggaggaagcggggctac1344argalatrpileglulysthrleuglugluglyarglysargglytyr435440445gtggaaaccctcttcggaagaaggcgctacgtgcccgacctcaacgcc1392valgluthrleupheglyargargargtyrvalproaspleuasnala450455460cgggtgaagagcgtcagggaggccgcggagcgcatggccttcaacatg1440argvallysservalargglualaalagluargmetalapheasnmet465470475480cccgtccagggcaccgccgccgacctcatgaagctcgccatggtgaag1488provalglnglythralaalaaspleumetlysleualametvallys485490495ctcttcccccgcctccgggagatgggggcccgcatgctcctccaggtc1536leupheproargleuargglumetglyalaargmetleuleuglnval500505510cacgacgagctcctcctggaggccccccaagcgcgggccgaggaggtg1584hisaspgluleuleuleuglualaproglnalaargalaglugluval515520525gcggctttggccaaggaggccatggagaaggcctatcccctcgccgtg1632alaalaleualalysglualametglulysalatyrproleualaval530535540cccctggaggtggaggtggggatgggggaggactggctttccgccaag1680proleugluvalgluvalglymetglygluasptrpleuseralalys545550555560ggttag1686gly 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