质粒名称: | pJSC038 - Bacterial expression plasmid for SpCas9∆REC3, HNH FRET variant |
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出品公司: | Addgene |
目录编号: | 101203 |
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/S355C/S867C/M1–N497,GGS,V713–D1368 |
物种来源: | Streptococcus pyogenes |
插入大小: | 951 bp |
基因突变: | C80S, C574S, S355C, S867C, 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: | pJSC038 - Bacterial expression plasmid for SpCas9∆REC3, HNH FRET variant was a gift from Jennifer Doudna & Keith Joung (Addgene plasmid # 101203 ; http://n2t.net/addgene:101203 ; RRID:Addgene_101203) |
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 |
pJSC038 - 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 pJSC038 - Bacterial expression plasmid for SpCas9∆REC3, HNH FRET variant 9437 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression plasmid for SpCas9∆REC3, HNH FRET variant. ACCESSION . VERSION . KEYWORDS pJSC038 - Bacterial expression plasmid for SpCas9∆REC3, HNH FR... 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 462..481 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 565..584 /label=T7 /note="T7 promoter, forward primer" promoter 565..583 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 672..694 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 714..743 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" CDS 753..1853 /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 1827..1850 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 1908..1928 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" primer_bind complement(5531..5549) /label=T7 Term /note="T7 terminator, reverse primer" terminator 5545..5592 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(5958..5986) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(6013..6031) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6099..6203 /gene="bla" /label=AmpR promoter CDS 6204..7064 /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(6422..6441) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7235..7823 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7724..7743 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7977..7994 /label=L4440 /note="L4440 vector, forward primer" misc_feature 8009..8148 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(8074..8093) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8234..8256 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(8250..8441) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc 61 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc 121 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag 181 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg 241 ttgaaggctc tcaagggcat cggtcgacgc tctcccttat gcgactcctg cattaggaag 301 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag 361 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa 421 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag 481 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg 541 atcgagatct cgatcccgcg aaattaatac gactcactat agggagacca caacggtttc 601 cctctagtgc cggctccgga gagctcttta attaagcggc cgccctgcag gactcgagtt 661 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgaaatct tctcaccatc 721 accatcacca tcaccatcac catggttctt ctatgaaaat cgaagaaggt aaactggtaa 781 tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga 841 aagataccgg aattaaagtc accgttgagc atccggataa actggaagag aaattcccac 901 aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg 961 gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc 1021 tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg 1081 ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct 1141 gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt 1201 tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt 1261 tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcaa 1321 aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg 1381 attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc 1441 cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga 1501 ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg 1561 ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag 1621 gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg 1681 aagagttggc gaaagatcca cgtattgccg ccactatgga aaacgcccag aaaggtgaaa 1741 tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca 1801 acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag actaattcga 1861 gctcgaacaa caacaacaat aacaataaca acaacctcgg gatcgaggaa aacctgtact 1921 tccaatccaa tgcaatggat aagaaatact caataggctt agatatcggc acaaatagcg 1981 tcggatgggc ggtgatcact gatgaatata aggttccgtc taaaaagttc aaggttctgg 2041 gaaatacaga ccgccacagt atcaaaaaaa atcttatagg ggctctttta tttgacagtg 2101 gagagacagc ggaagcgact cgtctcaaac ggacagctcg tagaaggtat acacgtcgga 2161 agaatcgtat ttcttatcta caggagattt tttcaaatga gatggcgaaa gtagatgata 2221 gtttctttca tcgacttgaa gagtcttttt tggtggaaga agacaagaag catgaacgtc 2281 atcctatttt tggaaatata gtagatgaag ttgcttatca tgagaaatat ccaactatct 2341 atcatctgcg aaaaaaattg gtagattcta ctgataaagc ggatttgcgc ttaatctatt 2401 tggccttagc gcatatgatt aagtttcgtg gtcatttttt gattgaggga gatttaaatc 2461 ctgataatag tgatgtggac aaactattta tccagttggt acaaacctac aatcaattat 2521 ttgaagaaaa ccctattaac gcaagtggag tagatgctaa agcgattctt tctgcacgat 2581 tgagtaaatc aagacgatta gaaaatctca ttgctcagct ccccggtgag aagaaaaatg 2641 gcttatttgg gaatctcatt gctttgtcat tgggtttgac ccctaatttt aaatcaaatt 2701 ttgatttggc agaagatgct aaattacagc tttcaaaaga tacttacgat gatgatttag 2761 ataatttatt ggcgcaaatt ggagatcaat atgctgattt gtttttggca gctaagaatt 2821 tatcagatgc tattttactt tcagatatcc taagagtaaa tactgaaata actaaggctc 2881 ccctatcagc ttcaatgatt aaacgctacg atgaacatca tcaagacttg actcttttaa 2941 aagctttagt tcgacaacaa cttccagaaa agtataaaga aatctttttt gatcaatgta 3001 aaaacggata tgcaggttat attgatgggg gagctagcca agaagaattt tataaattta 3061 tcaaaccaat tttagaaaaa atggatggta ctgaggaatt attggtgaaa ctaaatcgtg 3121 aagatttgct gcgcaagcaa cggacctttg acaacggctc tattccccat caaattcact 3181 tgggtgagct gcatgctatt ttgagaagac aagaagactt ttatccattt ttaaaagaca 3241 atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc ttattatgtt ggtccattgg 3301 cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc tgaagaaaca attaccccat 3361 ggaattttga agaagttgtc gataaaggtg cttcagctca atcatttatt gaacgcatga 3421 caaacggtgg aagtgtgtct ggacaaggcg atagtttaca tgaacatatt gcaaatttag 3481 ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt gatgaattgg 3541 tcaaagtaat ggggcggcat aagccagaaa atatagttat tgaaatggca cgtgaaaatc 3601 agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc gaagaaggta 3661 tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact caattgcaaa 3721 atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg gaccaagaat 3781 tagatattaa tcgtttaagt gattatgatg tcgatcacat tgttccacaa agtttcctta 3841 aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt ggtaaatgcg 3901 ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga caacttctaa 3961 acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa cgtggaggtt 4021 tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact cgccaaatca 4081 ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat gaaaatgata 4141 aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct gacttccgaa 4201 aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc catgatgcgt 4261 atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt gaatcggagt 4321 ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag tctgagcaag 4381 aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac ttcttcaaaa 4441 cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa actaatgggg 4501 aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc aaagtattgt 4561 ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga ttctccaagg 4621 agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa gactgggatc 4681 caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta gtggttgcta 4741 aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta gggatcacaa 4801 ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct aaaggatata 4861 aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt gagttagaaa 4921 acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat gagctggctc 4981 tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag ttgaagggta 5041 gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat tatttagatg 5101 agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat gccaatttag 5161 ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa caagcagaaa 5221 atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt aaatattttg 5281 atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat gccactctta 5341 tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag ctaggaggtg 5401 actaataaca ttggaagtgg ataacggatc cgcgatcgcg gcgcgccacc tggtggccgg 5461 ccggtaccac gcgtgcgctc tgatccggct gctaacaaag cccgaaagga agctgagttg 5521 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg 5581 aggggttttt tgctgaaagg aggaactata tccggatatc cacaggacgg gtgtggtcgc 5641 catgatcgcg tagtcgatag tggctccaag tagcgaagcg agcaggactg ggcggcggcc 5701 aaagcggtcg gacagtgctc cgagaacggg tgcgcataga aattgcatca acgcatatag 5761 cgctagcagc acgccatagt gactggcgat gctgtcggaa tggacgatat cccgcaagag 5821 gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag 5881 gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa 5941 ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt 6001 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 6061 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 6121 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 6181 aataacattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 6241 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 6301 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 6361 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 6421 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca 6481 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 6541 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 6601 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 6661 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 6721 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa 6781 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 6841 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 6901 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 6961 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 7021 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 7081 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 7141 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 7201 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 7261 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 7321 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 7381 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 7441 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 7501 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 7561 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 7621 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 7681 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 7741 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 7801 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 7861 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 7921 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 7981 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt 8041 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 8101 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg 8161 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 8221 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 8281 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc 8341 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata 8401 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg 8461 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg 8521 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta 8581 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca 8641 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg 8701 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt 8761 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt 8821 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac 8881 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc 8941 gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca 9001 ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg 9061 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg 9121 cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc 9181 tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg 9241 taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca 9301 gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga 9361 aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca 9421 tgccggcgat aatggcc //