质粒名称: | PX853 |
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出品公司: | 爱迪基因 |
目录编号: | 62885 |
存储实验室: | Feng Zhang |
载体骨架基本信息 |
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载体名称: | PX330 |
载体出品公司: | Zhang lab |
载体抗性: | 氨苄青霉素 |
空载体大小: | 4222bp |
完整质粒大小: | 6292 bp |
载体修饰: | 人细胞色素c氧化酶亚基VIII(human cytochrome C oxidase subunit VIII) |
载体类型: | 哺乳细胞表达 |
筛选标记: | 新霉素(G418) |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | SpCas9 (aa 2-573) |
物种来源: | -- |
插入大小: | 2073bp |
基因突变: | -- |
GenBank编号 | -- |
启动子 | -- |
标签/融合蛋白: | 人细胞色素c氧化酶亚基VIII(N端) |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | AgeI(未破坏) |
3' 克隆位点: | NotI (未破坏) |
5' 测序引物 | CMV-F |
3' 测序引物 | SV40pA-R |
文献引用方法 |
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材料和方法部分: | PX853 was a gift from Feng Zhang (Addgene plasmid # 62885 ; http://n2t.net/addgene:62885 ; RRID:Addgene_62885) |
参考文献部分: | A split-Cas9 architecture for inducible genome editing and transcription modulation. Zetsche B, Volz SE, Zhang F. Nat Biotechnol. 2015 Feb 2. doi: 10.1038/nbt.3149. 10.1038/nbt.3149 PubMed 25643054 |
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载体基本信息
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载体简介
载体序列
LOCUS PX853 6295 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION N-term SpCas9 piece of inducible split-5 (Cas9(N)-FRB-NES split-5).. ACCESSION . VERSION . KEYWORDS PX853 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6295) AUTHORS Zetsche B, Volz SE, Zhang F TITLE A split-Cas9 architecture for inducible genome editing and transcription modulation. JOURNAL Nat Biotechnol. 2015 Feb 2. doi: 10.1038/nbt.3149. PUBMED 25643054 REFERENCE 2 (bases 1 to 6295) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6295 /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 268..343 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 440..725 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 727..1004 /label=chicken beta-actin promoter intron 1005..1233 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 1278..2993 /codon_start=1 /product="N-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(N) /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE" CDS 3039..3308 /codon_start=1 /product="FKBP-rapamycin binding domain of human FRAP" /label=FRB /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ" primer_bind complement(3336..3353) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3342..3549 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 3558..3698 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3558..3687 /label=AAV2 ITR rep_origin 3773..4228 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3860..3879) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4070..4091 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4245..4264) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4364..4386 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4424..4442) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4510..4614 /gene="bla" /label=AmpR promoter CDS 4615..5475 /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(4833..4852) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5646..6234 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6135..6154 /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 ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc 1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atgctagatt 1261 tagctagctt aatcctcgac aagaagtaca gcatcggcct ggacatcggc accaactctg 1321 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg 1381 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg 1441 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga 1501 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca 1561 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc 1621 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct 1681 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc 1741 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc 1801 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt 1861 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac 1921 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg 1981 gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact 2041 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg 2101 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc 2161 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc 2221 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga 2281 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca 2341 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca 2401 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag 2461 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc 2521 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca 2581 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg 2641 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct 2701 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga 2761 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg 2821 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa 2881 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca 2941 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagggtggcg 3001 gtggctcggg tggcggtggc tcgggtggcg gtggctcgga gatgtggcac gagggcctgg 3061 aggaggccag cagactgtac ttcggcgaga gaaacgtgaa gggcatgttc gaggtgctgg 3121 agcccctgca cgccatgatg gagagaggcc cccagaccct gaaggagacc agcttcaacc 3181 aggcctacgg cagagacctg atggaggccc aggagtggtg cagaaagtac atgaagagcg 3241 gcaacgtgaa ggacctgacc caggcctggg acctgtacta ccacgtgttc agaagaatca 3301 gcaagcagta agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 3361 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 3421 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 3481 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca 3541 tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc 3601 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 3661 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct 3721 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc 3781 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 3841 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 3901 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 3961 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc 4021 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 4081 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt 4141 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 4201 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat 4261 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 4321 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 4381 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 4441 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 4501 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 4561 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 4621 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 4681 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 4741 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 4801 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 4861 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 4921 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 4981 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 5041 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 5101 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 5161 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 5221 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 5281 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt 5341 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 5401 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 5461 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 5521 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 5581 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 5641 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 5701 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 5761 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 5821 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 5881 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 5941 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 6001 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 6061 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 6121 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 6181 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 6241 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt //
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