| 质粒名称: | pJSC077 - Bacterial expression plasmid for SpCas9 + K855A, HNH FRET variant |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 101214 |
| 存储实验室: | Jennifer Doudna, Keith Joung |
|
载体骨架基本信息 |
|
| 载体名称: | pCT10 |
| 载体出品公司: | Doudna lab |
| 空载体大小: | -- |
| 完整质粒大小: | 10073 bp |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 氨苄青霉素，100μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | SpCas9 variant C80S/C574S/S355C/S867C/K855A |
| 物种来源: | Streptococcus pyogenes |
| 插入大小: | -- |
| 基因突变: | C80S, C574S, S355C, S867C and K855A |
| GenBank编号 | -- |
| 启动子: | T7 |
| 标签/融合蛋白: | 10xHis（主干上的N端子）MBP（主干上的N终端）TEV站点（主干上的N终端） |
| 克隆方法: | Gibson Cloning |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | T7 |
| 3’测序引物 | T7-term |
|
文献引用方法 |
|
| 材料和方法部分: | pJSC077 - Bacterial expression plasmid for SpCas9 + K855A, HNH FRET variant was a gift from Jennifer Doudna & Keith Joung (Addgene plasmid # 101214 ; http://n2t.net/addgene:101214 ; RRID:Addgene_101214) |
| 参考文献部分: | Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA. Nature. 2017 Sep 20. doi: 10.1038/nature24268. 10.1038/nature24268 PubMed 28931002 |
pJSC077 - Bacterial expression plasmid for SpCas9 + K855A, H
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pJSC077 - Bacterial expression plasmid for SpCas9 + K855A, H载体质粒基本信息
pJSC077 - Bacterial expression plasmid for SpCas9 + K855A, H质粒图谱载体图谱和pJSC077 - Bacterial expression plasmid for SpCas9 + K855A, H载体序列质粒序列多克隆位点信息
pJSC077 - Bacterial expression plasmid for SpCas9 + K855A, H质粒载体简介
pJSC077 - Bacterial expression plasmid for SpCas9 + K855A, H质粒序列载体序列
LOCUS pJSC077 - Bacterial expression plasmid for SpCas9 + K855A, HNH FRET variant 10073 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Bacterial expression plasmid for SpCas9 + K855A, HNH FRET variant.
ACCESSION .
VERSION .
KEYWORDS pJSC077 - Bacterial expression plasmid for SpCas9 + K855A, HNH...
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10073)
AUTHORS Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington
LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA
TITLE Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.
JOURNAL Nature. 2017 Sep 20. doi: 10.1038/nature24268.
PUBMED 28931002
REFERENCE 2 (bases 1 to 10073)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..10073
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 543..562
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 646..665
/label=T7
/note="T7 promoter, forward primer"
promoter 646..664
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
RBS 753..775
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 795..824
/codon_start=1
/product="10xHis affinity tag"
/label=10xHis
/translation="HHHHHHHHHH"
CDS 834..1934
/codon_start=1
/gene="malE (mutated)"
/product="maltose binding protein from E. coli"
/label=MBP
/note="This version of the gene does not encode a signal
sequence, so MBP will remain in the cytosol."
/translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
WYAVRTAVINAASGRQTVDEALKDAQT"
primer_bind 1908..1931
/label=MBP-F
/note="Maltose binding protein, forward primer"
CDS 1989..2009
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
CDS 2016..6122
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRISYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQCKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIESFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNAVLTRSDKNRGKCDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
primer_bind complement(6248..6266)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 6262..6309
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(6675..6703)
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
primer_bind complement(6730..6748)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 6816..6920
/gene="bla"
/label=AmpR promoter
CDS 6921..7781
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(7139..7158)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 7952..8540
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8441..8460
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 8694..8711
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 8726..8865
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(8791..8810)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8951..8973
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(8967..9158)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
ORIGIN
1 gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc
61 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
121 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
181 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
241 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
301 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgacg ctctccctta
361 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
421 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
481 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
541 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
601 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
661 tagggagacc acaacggttt ccctctagtg ccggctccgg agagctcttt aattaagcgg
721 ccgccctgca ggactcgagt tctagaaata attttgttta actttaagaa ggagatatac
781 atatgaaatc ttctcaccat caccatcacc atcaccatca ccatggttct tctatgaaaa
841 tcgaagaagg taaactggta atctggatta acggcgataa aggctataac ggtctcgctg
901 aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata
961 aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac attatcttct
1021 gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg
1081 acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca
1141 agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc
1201 tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga
1261 aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg
1321 ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg
1381 gcgtggataa cgctggcgca aaagcgggtc tgaccttcct ggttgacctg attaaaaaca
1441 aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa
1501 cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt
1561 atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc
1621 tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa
1681 actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg
1741 tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccactatgg
1801 aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg
1861 ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga
1921 aagacgcgca gactaattcg agctcgaaca acaacaacaa taacaataac aacaacctcg
1981 ggatcgagga aaacctgtac ttccaatcca atgcaatgga taagaaatac tcaataggct
2041 tagatatcgg cacaaatagc gtcggatggg cggtgatcac tgatgaatat aaggttccgt
2101 ctaaaaagtt caaggttctg ggaaatacag accgccacag tatcaaaaaa aatcttatag
2161 gggctctttt atttgacagt ggagagacag cggaagcgac tcgtctcaaa cggacagctc
2221 gtagaaggta tacacgtcgg aagaatcgta tttcttatct acaggagatt ttttcaaatg
2281 agatggcgaa agtagatgat agtttctttc atcgacttga agagtctttt ttggtggaag
2341 aagacaagaa gcatgaacgt catcctattt ttggaaatat agtagatgaa gttgcttatc
2401 atgagaaata tccaactatc tatcatctgc gaaaaaaatt ggtagattct actgataaag
2461 cggatttgcg cttaatctat ttggccttag cgcatatgat taagtttcgt ggtcattttt
2521 tgattgaggg agatttaaat cctgataata gtgatgtgga caaactattt atccagttgg
2581 tacaaaccta caatcaatta tttgaagaaa accctattaa cgcaagtgga gtagatgcta
2641 aagcgattct ttctgcacga ttgagtaaat caagacgatt agaaaatctc attgctcagc
2701 tccccggtga gaagaaaaat ggcttatttg ggaatctcat tgctttgtca ttgggtttga
2761 cccctaattt taaatcaaat tttgatttgg cagaagatgc taaattacag ctttcaaaag
2821 atacttacga tgatgattta gataatttat tggcgcaaat tggagatcaa tatgctgatt
2881 tgtttttggc agctaagaat ttatcagatg ctattttact ttcagatatc ctaagagtaa
2941 atactgaaat aactaaggct cccctatcag cttcaatgat taaacgctac gatgaacatc
3001 atcaagactt gactctttta aaagctttag ttcgacaaca acttccagaa aagtataaag
3061 aaatcttttt tgatcaatgt aaaaacggat atgcaggtta tattgatggg ggagctagcc
3121 aagaagaatt ttataaattt atcaaaccaa ttttagaaaa aatggatggt actgaggaat
3181 tattggtgaa actaaatcgt gaagatttgc tgcgcaagca acggaccttt gacaacggct
3241 ctattcccca tcaaattcac ttgggtgagc tgcatgctat tttgagaaga caagaagact
3301 tttatccatt tttaaaagac aatcgtgaga agattgaaaa aatcttgact tttcgaattc
3361 cttattatgt tggtccattg gcgcgtggca atagtcgttt tgcatggatg actcggaagt
3421 ctgaagaaac aattacccca tggaattttg aagaagttgt cgataaaggt gcttcagctc
3481 aatcatttat tgaacgcatg acaaactttg ataaaaatct tccaaatgaa aaagtactac
3541 caaaacatag tttgctttat gagtatttta cggtttataa cgaattgaca aaggtcaaat
3601 atgttactga aggaatgcga aaaccagcat ttctttcagg tgaacagaag aaagccattg
3661 ttgatttact cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa gaagattatt
3721 tcaaaaaaat agaatctttt gatagtgttg aaatttcagg agttgaagat agatttaatg
3781 cttcattagg tacctaccat gatttgctaa aaattattaa agataaagat tttttggata
3841 atgaagaaaa tgaagatatc ttagaggata ttgttttaac attgacctta tttgaagata
3901 gggagatgat tgaggaaaga cttaaaacat atgctcacct ctttgatgat aaggtgatga
3961 aacagcttaa acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa ttgattaatg
4021 gtattaggga taagcaatct ggcaaaacaa tattagattt tttgaaatca gatggttttg
4081 ccaatcgcaa ttttatgcag ctgatccatg atgatagttt gacatttaaa gaagacattc
4141 aaaaagcaca agtgtctgga caaggcgata gtttacatga acatattgca aatttagctg
4201 gtagccctgc tattaaaaaa ggtattttac agactgtaaa agttgttgat gaattggtca
4261 aagtaatggg gcggcataag ccagaaaata tcgttattga aatggcacgt gaaaatcaga
4321 caactcaaaa gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca
4381 aagaattagg aagtcagatt cttaaagagc atcctgttga aaatactcaa ttgcaaaatg
4441 aaaagctcta tctctattat ctccaaaatg gaagagacat gtatgtggac caagaattag
4501 atattaatcg tttaagtgat tatgatgtcg atcacattgt tccacaaagt ttccttaaag
4561 acgattcaat agacaatgct gtcttaacgc gttctgataa aaatcgtggt aaatgcgata
4621 acgttccaag tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg
4681 ccaagttaat cactcaacgt aagtttgata atttaacgaa agctgaacgt ggaggtttga
4741 gtgaacttga taaagctggt tttatcaaac gccaattggt tgaaactcgc caaatcacta
4801 agcatgtggc acaaattttg gatagtcgca tgaatactaa atacgatgaa aatgataaac
4861 ttattcgaga ggttaaagtg attaccttaa aatctaaatt agtttctgac ttccgaaaag
4921 atttccaatt ctataaagta cgtgagatta acaattacca tcatgcccat gatgcgtatc
4981 taaatgccgt cgttggaact gctttgatta agaaatatcc aaaacttgaa tcggagtttg
5041 tctatggtga ttataaagtt tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa
5101 taggcaaagc aaccgcaaaa tatttctttt actctaatat catgaacttc ttcaaaacag
5161 aaattacact tgcaaatgga gagattcgca aacgccctct aatcgaaact aatggggaaa
5221 ctggagaaat tgtctgggat aaagggcgag attttgccac agtgcgcaaa gtattgtcca
5281 tgccccaagt caatattgtc aagaaaacag aagtacagac aggcggattc tccaaggagt
5341 caattttacc aaaaagaaat tcggacaagc ttattgctcg taaaaaagac tgggatccaa
5401 aaaaatatgg tggttttgat agtccaacgg tagcttattc agtcctagtg gttgctaagg
5461 tggaaaaagg gaaatcgaag aagttaaaat ccgttaaaga gttactaggg atcacaatta
5521 tggaaagaag ttcctttgaa aaaaatccga ttgacttttt agaagctaaa ggatataagg
5581 aagttaaaaa agacttaatc attaaactac ctaaatatag tctttttgag ttagaaaacg
5641 gtcgtaaacg gatgctggct agtgccggag aattacaaaa aggaaatgag ctggctctgc
5701 caagcaaata tgtgaatttt ttatatttag ctagtcatta tgaaaagttg aagggtagtc
5761 cagaagataa cgaacaaaaa caattgtttg tggagcagca taagcattat ttagatgaga
5821 ttattgagca aatcagtgaa ttttctaagc gtgttatttt agcagatgcc aatttagata
5881 aagttcttag tgcatataac aaacatagag acaaaccaat acgtgaacaa gcagaaaata
5941 ttattcattt atttacgttg acgaatcttg gagctcccgc tgcttttaaa tattttgata
6001 caacaattga tcgtaaacga tatacgtcta caaaagaagt tttagatgcc actcttatcc
6061 atcaatccat cactggtctt tatgaaacac gcattgattt gagtcagcta ggaggtgact
6121 aataacattg gaagtggata acggatccgc gatcgcggcg cgccacctgg tggccggccg
6181 gtaccacgcg tgcgctctga tccggctgct aacaaagccc gaaaggaagc tgagttggct
6241 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
6301 ggttttttgc tgaaaggagg aactatatcc ggatatccac aggacgggtg tggtcgccat
6361 gatcgcgtag tcgatagtgg ctccaagtag cgaagcgagc aggactgggc ggcggccaaa
6421 gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg catatagcgc
6481 tagcagcacg ccatagtgac tggcgatgct gtcggaatgg acgatatccc gcaagaggcc
6541 cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat
6601 gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg
6661 tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag aattcttgaa
6721 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
6781 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
6841 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
6901 aacattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
6961 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
7021 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
7081 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
7141 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
7201 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
7261 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
7321 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
7381 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
7441 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
7501 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
7561 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
7621 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
7681 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
7741 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
7801 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
7861 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
7921 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
7981 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
8041 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
8101 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
8161 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
8221 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
8281 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
8341 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
8401 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
8461 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
8521 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
8581 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
8641 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
8701 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
8761 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
8821 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
8881 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
8941 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
9001 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
9061 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
9121 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
9181 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
9241 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
9301 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
9361 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
9421 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
9481 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
9541 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
9601 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
9661 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
9721 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
9781 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
9841 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
9901 ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa
9961 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
10021 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atg
//
