质粒名称: | pJSC057 - Bacterial expression plasmid for SpCas9∆REC3, REC2 FRET variant |
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出品公司: | Addgene |
目录编号: | 101204 |
存储实验室: | Jennifer Doudna, Keith Joung |
载体骨架基本信息 |
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载体名称: | pCT10 |
载体出品公司: | Doudna lab |
空载体大小: | -- |
完整质粒大小: | 9437 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 |
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插入基因名称: | SpCas9 variant C80S/C574S/E60C/D273C/M1–N497,GGS,V713–D1368 |
物种来源: | Streptococcus pyogenes |
插入大小: | -- |
基因突变: | C80S, C574S, E60C, D273C, M1-N497, GGS, V713-D1368 |
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 |
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材料和方法部分: | pJSC057 - Bacterial expression plasmid for SpCas9∆REC3, REC2 FRET variant was a gift from Jennifer Doudna & Keith Joung (Addgene plasmid # 101204 ; http://n2t.net/addgene:101204 ; RRID:Addgene_101204) |
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 |
pJSC057 - Bacterial expression plasmid for SpCas9∆REC3
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pJSC057 - Bacterial expression plasmid for SpCas9∆REC3, REC2 FRET variant 9437 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression plasmid for SpCas9∆REC3, REC2 FRET variant. ACCESSION . VERSION . KEYWORDS pJSC057 - Bacterial expression plasmid for SpCas9∆REC3, REC2 F... SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9437) 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 9437) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..9437 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 139..158 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 242..261 /label=T7 /note="T7 promoter, forward primer" promoter 242..260 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 349..371 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 391..420 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" CDS 430..1530 /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 1504..1527 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 1585..1605 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" primer_bind complement(5208..5226) /label=T7 Term /note="T7 terminator, reverse primer" terminator 5222..5269 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(5635..5663) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(5690..5708) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5776..5880 /gene="bla" /label=AmpR promoter CDS 5881..6741 /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(6099..6118) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6912..7500 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7401..7420 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7654..7671 /label=L4440 /note="L4440 vector, forward primer" misc_feature 7686..7825 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(7751..7770) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7911..7933 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(7927..8118) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc 61 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg 121 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc 181 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa 241 ttaatacgac tcactatagg gagaccacaa cggtttccct ctagtgccgg ctccggagag 301 ctctttaatt aagcggccgc cctgcaggac tcgagttcta gaaataattt tgtttaactt 361 taagaaggag atatacatat gaaatcttct caccatcacc atcaccatca ccatcaccat 421 ggttcttcta tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc 481 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc 541 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc 601 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg 661 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc 721 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt 781 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat 841 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc 901 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac 961 gacattaaag acgtgggcgt ggataacgct ggcgcaaaag cgggtctgac cttcctggtt 1021 gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc 1081 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac 1141 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa 1201 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca 1261 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac 1321 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt 1381 attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg 1441 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact 1501 gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacaataac 1561 aataacaaca acctcgggat cgaggaaaac ctgtacttcc aatccaatgc aatggataag 1621 aaatactcaa taggcttaga tatcggcaca aatagcgtcg gatgggcggt gatcactgat 1681 gaatataagg ttccgtctaa aaagttcaag gttctgggaa atacagaccg ccacagtatc 1741 aaaaaaaatc ttataggggc tcttttattt gacagtggag agacagcgtg tgcgactcgt 1801 ctcaaacgga cagctcgtag aaggtataca cgtcggaaga atcgtatttc ttatctacag 1861 gagatttttt caaatgagat ggcgaaagta gatgatagtt tctttcatcg acttgaagag 1921 tcttttttgg tggaagaaga caagaagcat gaacgtcatc ctatttttgg aaatatagta 1981 gatgaagttg cttatcatga gaaatatcca actatctatc atctgcgaaa aaaattggta 2041 gattctactg ataaagcgga tttgcgctta atctatttgg ccttagcgca tatgattaag 2101 tttcgtggtc attttttgat tgagggagat ttaaatcctg ataatagtga tgtggacaaa 2161 ctatttatcc agttggtaca aacctacaat caattatttg aagaaaaccc tattaacgca 2221 agtggagtag atgctaaagc gattctttct gcacgattga gtaaatcaag acgattagaa 2281 aatctcattg ctcagctccc cggtgagaag aaaaatggct tatttgggaa tctcattgct 2341 ttgtcattgg gtttgacccc taattttaaa tcaaattttg atttggcaga agatgctaaa 2401 ttacagcttt caaaagatac ttacgattgt gatttagata atttattggc gcaaattgga 2461 gatcaatatg ctgatttgtt tttggcagct aagaatttat cagatgctat tttactttca 2521 gatatcctaa gagtaaatac tgaaataact aaggctcccc tatcagcttc aatgattaaa 2581 cgctacgatg aacatcatca agacttgact cttttaaaag ctttagttcg acaacaactt 2641 ccagaaaagt ataaagaaat cttttttgat caatcaaaaa acggatatgc aggttatatt 2701 gatgggggag ctagccaaga agaattttat aaatttatca aaccaatttt agaaaaaatg 2761 gatggtactg aggaattatt ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg 2821 acctttgaca acggctctat tccccatcaa attcacttgg gtgagctgca tgctattttg 2881 agaagacaag aagactttta tccattttta aaagacaatc gtgagaagat tgaaaaaatc 2941 ttgacttttc gaattcctta ttatgttggt ccattggcgc gtggcaatag tcgttttgca 3001 tggatgactc ggaagtctga agaaacaatt accccatgga attttgaaga agttgtcgat 3061 aaaggtgctt cagctcaatc atttattgaa cgcatgacaa acggtggaag tgtgtctgga 3121 caaggcgata gtttacatga acatattgca aatttagctg gtagccctgc tattaaaaaa 3181 ggtattttac agactgtaaa agttgttgat gaattggtca aagtaatggg gcggcataag 3241 ccagaaaata tcgttattga aatggcacgt gaaaatcaga caactcaaaa gggccagaaa 3301 aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca aagaattagg aagtcagatt 3361 cttaaagagc atcctgttga aaatactcaa ttgcaaaatg aaaagctcta tctctattat 3421 ctccaaaatg gaagagacat gtatgtggac caagaattag atattaatcg tttaagtgat 3481 tatgatgtcg atcacattgt tccacaaagt ttccttaaag acgattcaat agacaataag 3541 gtcttaacgc gttctgataa aaatcgtggt aaatcggata acgttccaag tgaagaagta 3601 gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg ccaagttaat cactcaacgt 3661 aagtttgata atttaacgaa agctgaacgt ggaggtttga gtgaacttga taaagctggt 3721 tttatcaaac gccaattggt tgaaactcgc caaatcacta agcatgtggc acaaattttg 3781 gatagtcgca tgaatactaa atacgatgaa aatgataaac ttattcgaga ggttaaagtg 3841 attaccttaa aatctaaatt agtttctgac ttccgaaaag atttccaatt ctataaagta 3901 cgtgagatta acaattacca tcatgcccat gatgcgtatc taaatgccgt cgttggaact 3961 gctttgatta agaaatatcc aaaacttgaa tcggagtttg tctatggtga ttataaagtt 4021 tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa taggcaaagc aaccgcaaaa 4081 tatttctttt actctaatat catgaacttc ttcaaaacag aaattacact tgcaaatgga 4141 gagattcgca aacgccctct aatcgaaact aatggggaaa ctggagaaat tgtctgggat 4201 aaagggcgag attttgccac agtgcgcaaa gtattgtcca tgccccaagt caatattgtc 4261 aagaaaacag aagtacagac aggcggattc tccaaggagt caattttacc aaaaagaaat 4321 tcggacaagc ttattgctcg taaaaaagac tgggatccaa aaaaatatgg tggttttgat 4381 agtccaacgg tagcttattc agtcctagtg gttgctaagg tggaaaaagg gaaatcgaag 4441 aagttaaaat ccgttaaaga gttactaggg atcacaatta tggaaagaag ttcctttgaa 4501 aaaaatccga ttgacttttt agaagctaaa ggatataagg aagttaaaaa agacttaatc 4561 attaaactac ctaaatatag tctttttgag ttagaaaacg gtcgtaaacg gatgctggct 4621 agtgccggag aattacaaaa aggaaatgag ctggctctgc caagcaaata tgtgaatttt 4681 ttatatttag ctagtcatta tgaaaagttg aagggtagtc cagaagataa cgaacaaaaa 4741 caattgtttg tggagcagca taagcattat ttagatgaga ttattgagca aatcagtgaa 4801 ttttctaagc gtgttatttt agcagatgcc aatttagata aagttcttag tgcatataac 4861 aaacatagag acaaaccaat acgtgaacaa gcagaaaata ttattcattt atttacgttg 4921 acgaatcttg gagctcccgc tgcttttaaa tattttgata caacaattga tcgtaaacga 4981 tatacgtcta caaaagaagt tttagatgcc actcttatcc atcaatccat cactggtctt 5041 tatgaaacac gcattgattt gagtcagcta ggaggtgact aataacattg gaagtggata 5101 acggatccgc gatcgcggcg cgccacctgg tggccggccg gtaccacgcg tgcgcgctga 5161 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata 5221 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg 5281 aactatatcc ggatatccac aggacgggtg tggtcgccat gatcgcgtag tcgatagtgg 5341 ctccaagtag cgaagcgagc aggactgggc ggcggccaaa gcggtcggac agtgctccga 5401 gaacgggtgc gcatagaaat tgcatcaacg catatagcgc tagcagcacg ccatagtgac 5461 tggcgatgct gtcggaatgg acgatatccc gcaagaggcc cggcagtacc ggcataacca 5521 agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag 5581 atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa 5641 gcttatcgat gataagctgt caaacatgag aattcttgaa gacgaaaggg cctcgtgata 5701 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact 5761 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 5821 tatccgctca tgagacaata accctgataa atgcttcaat aacattgaaa aaggaagagt 5881 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 5941 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 6001 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 6061 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 6121 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 6181 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 6241 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 6301 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 6361 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 6421 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 6481 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 6541 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 6601 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 6661 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 6721 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 6781 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 6841 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 6901 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 6961 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 7021 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 7081 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 7141 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 7201 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 7261 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 7321 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 7381 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 7441 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 7501 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 7561 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 7621 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 7681 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt 7741 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc 7801 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg 7861 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 7921 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc 7981 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt 8041 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt 8101 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat 8161 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg 8221 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa 8281 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag 8341 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt 8401 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga 8461 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc 8521 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac 8581 ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg agatggcgga 8641 cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc gcaagaattg 8701 attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc ttccattcag 8761 gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg 8821 gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc ataaatcgcc 8881 gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag cgatccttga 8941 agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa cgcgggcatc 9001 ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc tcgcgtcgcg 9061 aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc 9121 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt 9181 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg 9241 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc 9301 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag 9361 gctctcaagg gcatcggtcg acgctctccc ttatgcgact cctgcattag gaagcagccc 9421 agtagtaggt tgaggcc //