质粒名称: | xCas12i |
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出品公司: | Addgene |
目录编号: | 195333 |
存储实验室: | Yingsi Zhou |
载体骨架基本信息 |
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载体名称: | px330 |
载体出品公司: | Feng Zhang (Addgene plasmid # 42230) |
完整质粒大小: | 9321bp |
载体类型: | 哺乳细胞表达 |
载体抗性: | 氨苄霉素 |
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: | human codon-optimized xCas12i |
物种来源: | Synthetic |
插入大小: | 951 bp |
启动子: | CBh, CMV, hU6 |
标签/融合蛋白: | 3xFlag (N terminal on insert) |
克隆方法: | Gibson Cloning |
文献引用方法 |
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材料和方法部分: | xCas12i was a gift from Yingsi Zhou (Addgene plasmid # 195333 ; http://n2t.net/addgene:195333 ; RRID:Addgene_195333) |
参考文献部分: | An engineered xCas12i with high activity, high specificity, and broad PAM range. Zhang H, Kong X, Xue M, Hu J, Wang Z, Wei Y, Wang H, Zhou J, Zhang W, Xu M, Shen X, Yin F, Ai Z, Huang G, Xia J, Song X, Li H, Yuan Y, Li J, Zhong N, Zhang M, Zhou Y, Yang H. Protein Cell. 2023 Jun 28;14(7):538-543. doi: 10.1093/procel/pwac052. 10.1093/procel/pwac052 PubMed 37378659 |
买家导航
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 xCas12i 9321 bp ds-DNA circular SYN 30-JAN-2023 DEFINITION vector for encoding a human codon-optimized xCas12i driven by CBh promoter, guide RNAs compatible with xCas12i driven by hU6, and mCherry driven by CMV promoter. ACCESSION . VERSION . KEYWORDS xCas12i SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9321) AUTHORS Zhang H, Kong X, Xue M, Hu J, Wang Z, Wei Y, Wang H, Zhou J, Zhang W, Xu M, Shen X, Yin F, Ai Z, Huang G, Xia J, Song X, Li H, Yuan Y, Li J, Zhong N, Zhang M, Zhou Y, Yang H TITLE An engineered xCas12i with high activity, high specificity and broad PAM range JOURNAL REFERENCE 2 (bases 1 to 9321) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 30, 2023 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..9321 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 299..374 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 471..756 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 758..1035 /label=chicken beta-actin promoter intron 1036..1263 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1275..1284 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1284..1349 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1356..1376 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4641..4688 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(4716..4733) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4722..4929 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" enhancer 4998..5301 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 5302..5505 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 5456..5476 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 5534..6244 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(5679..5697) /label=mCherry-R /note="mCherry, reverse primer" rep_origin 6799..7254 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6886..6905) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7096..7117 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7271..7290) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7390..7412 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7450..7468) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7536..7640 /gene="bla" /label=AmpR promoter CDS 7641..8501 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(7859..7878) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8672..9260 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9161..9180 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg agaaatgtgt ccccagttga cacagagacc gagaggtctc gtttttttgt 301 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg 361 caccgagtcg gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc 421 cgtttttagc gcgtgcgcca attctgcaga caaatggctc tagaggtacc cgttacataa 481 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 541 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 601 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 661 ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg 721 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc 781 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa 841 ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg cggggcgggg 901 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc 961 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga 1021 agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc 1081 gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg 1141 gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt taagggatgg 1201 ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat cacttttttt 1261 caggttggac cggtgccacc atggactata aggaccacga cggagactac aaggatcatg 1321 atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg aaggtcggta 1381 tccacggagt cccagcagcc atgagttctg atgtggtgcg gccttataac acaaagctgc 1441 tcccagataa cagaaagcac aatatgttcc tgcagacctt caagcggctg aacagcatct 1501 ctctgaacca cttcgacctg ctgatctgcc tgtacgctgc aatcaccaac aagaaggccg 1561 aggaatacaa gtctgaaaag gaagcccacg tgaccgccga tagcctgtgt gccatcaatt 1621 ggttcagacc catgagcaag agatacagca aatacgccac caccaccttc aacatgttag 1681 aactgtttaa ggagtacagc ggccacgagc ctgatgccta ttccaagaac tacctgatga 1741 gcaatatcga cagcgacaga ttcgtgtggg tggattgtag gaagttcgct aaggactttg 1801 cctatcagat ggaactgggt ttccacgagt tcaccgtgtt ggccgaaacc ctgctggcta 1861 attctatcct ggtgctgaac gagagcacca aggccaattg ggcttgggga accgtgtctg 1921 ccctgtacgg cggcggagat aaggaggaca gcacactgaa gagcaagatt ctgctggcct 1981 tcgtggacgc cctgaacaac cacgagctga aaacaaagag agaaatcttg aatcaagtgt 2041 gtgaatctct gaaataccag agctaccagg acatgtacgt ggattttaga agcgtggttg 2101 acgaaaacgg caacaagaag tctcctaacg gctctatgcc tatcgtgacc aagttcgaga 2161 cagacgacct gatcagcgac aaccaaagaa aggccatgat cagcaacttc actaagaacg 2221 ccgctgccaa ggcagctaag aaacctatcc cttacttgga ccgcctgaag gagcacatgg 2281 tgtccctgtg cgacgagtac aatgtgtatg cctgggccgc ggccatcaca aacagcaacg 2341 ccgacgtgac cgcccggaat accagaaacc tgacattcat cggcgaacag aacagcagac 2401 gaaaggaact gagcgtgctg cagacaacaa ccaacgagaa ggctaaggac atcctgaaca 2461 agatcaacga caacctgatt caggaggtgc ggtacacccc tgcccctaag cacctgggca 2521 gagatctggc caacctgttt gatacactga aggaaaagga catcaacaac atcgagaacg 2581 aagaagagaa acagaacgtg atcaatgact gtatcgagca gtacgtggac gattgcagaa 2641 gcctcaaccg gaaccccatc gcagccctcc tgaagcacat ctctaggtac tacgaggatt 2701 tcagcgccaa gaatttcctg gacggcgcca agctgaacgt gctgactgag gtggtgaacc 2761 ggcagaaggc ccaccccacc atctggagcg agaaggctta cacctggatc agcaagttcg 2821 acaagaaccg gagacaggcc aacagcagcc tggtcggatg ggttgtgccc cccgaggagg 2881 tgcacaagga gaaaatcgcc ggacagcaga gcatgatgtg ggtgaccctc accctgctgg 2941 acgacggcaa gtgggtcaaa catcacatcc ccttcagcga cagcagatac tacagcgaag 3001 tgtacgccta caaccctaat ctgccttatc tggacggagg catcccaaga cagagcaagt 3061 tcggcaacaa accaacaacc aacctgacag ccgagtccca ggccctcctg gctaattcta 3121 agtacaagaa agccaacaag agcttcctgc gggctaaaga gaatgccaca cacaacgtgc 3181 gggtgtcccc taacacctct ctgtgcatta gactgctgaa ggacagcgcc ggaaaccaga 3241 tgttcgacaa aatcggcaac gtgctcttcg gcatgcagat caaccacaag atcaccgtgg 3301 gaaaacctaa ctacaagatc gaggtgggcg acagattcct gggcttcgat cagaaccaga 3361 gcgagaacca cacctacgcc gtgctgcaga gagtgtccga gagcagtcac gacacccacc 3421 actttaacgg ctgggacgtg aaggtgctgg aaaagggcaa agtgaccagc gatgtgatcg 3481 tgcgggacga ggtctacgac caactgtctt acgagggcgt cccctacgat agcagcaagt 3541 tcgccgagtg gcgggacaag cgcagaagat ttgtgcttga gaacctgagc atccagctgg 3601 aagagggcaa gaccttcctg acagagttcg acaagctgaa taaggacagc ctgtaccgct 3661 ggaacatgaa ctacctgaaa ctgctgagaa aggccatccg ggccggaggc aaagagttcg 3721 ccaagatcgc taagacagag atcttcgagc tggcggtgga aagattcggc cctattaacc 3781 tgggcagcct gtcccagatc agccttaaga tgattgcctc ctttaagggc gtggtccagt 3841 cctacttctc cgtgagcggc tgcgtggatg atgcctccaa aaaggcccat gattctatgc 3901 tgttcacatt tatgtgcgcc gccgaagaaa agcggaccaa caagagagaa gaaaagacca 3961 acagagccgc cagctttatc ctgcaaaaag cctacctgca tggctgcaag atgatcgtgt 4021 gcgaggacga ccttcctgtg gccgacggca agacaggcaa agcccagaat gccgaccgga 4081 tggactggtg cgccagagcc ctggccaaga aggtgaacga cggctgtgtt gccatgagca 4141 tctgctacag agctatccct gcctacatga gcagccacca ggaccccttt gtgcacatgc 4201 aggataagaa aaccagcgtg ctgcggccta gattcatgga agttaataag gatagcatca 4261 gagactacca cgtggcgggc ctgagaagaa tgctgaacag caagagtgac gctggcacca 4321 gtgtttatta ccggcaagct gccctgcatt tctgcgaagc cctgggcgtg agccctgaac 4381 tggtgaaaaa caagaaaacc cacgccgccg aactgggcaa gcacatgggc agcgctatgc 4441 tgatgccctg gagaggcggt agagtgtaca tcgccagcaa aaagctgacc tccgatgcca 4501 aatcagtgaa gtactgcggc gaggatatgt ggcagtacca cgccgatgag atcgccgctg 4561 ttaacatcgc catgtatgag gtgtgctgcc agaccggcgc tttcggaaag aaacagaaaa 4621 aatcggacga gctgcctgga aaaaggccgg cggccacgaa gaaggccggc caggcaaaga 4681 agaagaagta agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 4741 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 4801 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 4861 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca 4921 tgctggggag cggccgctag ttattaatag taatcaatta cggggtcatt agttcatagc 4981 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 5041 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 5101 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 5161 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 5221 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 5281 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 5341 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 5401 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 5461 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt 5521 cagatccgct agcatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt 5581 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg 5641 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg 5701 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc 5761 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt 5821 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc 5881 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc 5941 cgacggcccc gtaatgcaga agaaaaccat gggctgggag gcctcctccg agcggatgta 6001 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg 6061 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg 6121 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt 6181 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa 6241 gtaaggatcc gggatgcaga aattgatgat ctattaaaca ataaagatgt ccactaaaat 6301 ggaagttttt cctgtcatac tttgttaaga agggtgagaa cagagtacct acattttgaa 6361 tggaaggatt ggagctacgg gggtgggggt ggggtgggat tagataaatg cctgctcttt 6421 actgaaggct ctttactatt gctttatgat aatgtttcat agttggatat cataatttaa 6481 acaagcaaaa ccaaattaag ggccagctca ttcctcccac tcatgatcta tagatctata 6541 gatctctcgt gggatcattg tttttctctt gattcccact ttgtggttct aagtactgtg 6601 gtttccaaat gtgtcagttt catagcctga agaacgagat cagcagcctc tgttccacat 6661 acacttcatt ctcagtattg ttttgccaag ttctaattcc atcagaagct ggtcgacctg 6721 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata 6781 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 6841 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct 6901 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 6961 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg 7021 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 7081 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg 7141 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc 7201 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt 7261 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 7321 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 7381 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 7441 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 7501 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 7561 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 7621 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 7681 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 7741 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 7801 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 7861 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 7921 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 7981 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 8041 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 8101 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 8161 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 8221 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 8281 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 8341 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct 8401 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 8461 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 8521 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 8581 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 8641 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 8701 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 8761 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 8821 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 8881 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 8941 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 9001 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 9061 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 9121 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 9181 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 9241 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 9301 gctggccttt tgctcacatg t //