质粒名称: | AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR |
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出品公司: | 爱迪基因 |
目录编号: | 60229 |
存储实验室: | Feng Zhang |
载体骨架基本信息 |
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载体名称: | AAV |
载体抗性: | 氨苄青霉素 |
空载体大小: | 2597 bp |
完整质粒大小: | 6394 bp |
载体类型: | 哺乳动物表达,小鼠靶向,AAV,Cre/Lox,CRISPR |
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: | sgRNA,single guide RNA,guide RNA,gRNA |
插入大小: | 100 bp |
启动子 | U6 |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | MluI(未破坏) |
3' 克隆位点: | XbaI (未破坏) |
5' 测序引物 | GAGGGCCTATTTCCCATGATTC |
文献引用方法 |
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材料和方法部分: | AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR was a gift from Feng Zhang (Addgene plasmid # 60229 ; http://n2t.net/addgene:60229 ; RRID:Addgene_60229) |
参考文献部分: | CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F. Cell. 2014 Sep 24. pii: S0092-8674(14)01163-5. doi: 10.1016/j.cell.2014.09.014. 10.1016/j.cell.2014.09.014 PubMed 25263330 |
AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR
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 AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR 6362 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Expresses Cre recombinase from the Cbh promoter and one U6-driven sgRNA. AAV backbone with SapI spacer for sgRNA cloning.. ACCESSION . VERSION . KEYWORDS AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6362) AUTHORS Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F TITLE CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. JOURNAL Cell. 2014 Sep 24. pii: S0092-8674(14)01163-5. doi: 10.1016/j.cell.2014.09.014. PUBMED 25263330 REFERENCE 2 (bases 1 to 6362) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6362 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 145..385 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 145..165 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 316..335 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 412..487 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 577..862 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 864..1139 /label=chicken beta-actin promoter intron 1140..1367 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 1388..1408 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2435..2461 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" misc_feature 2489..3077 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2542..2562) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2960..2971) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3109..3585 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3308..3327) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 3625..3765 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3625..3754 /label=AAV2 ITR rep_origin 3840..4295 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3927..3946) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4137..4158 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4312..4331) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4431..4453 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4491..4509) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4577..4681 /gene="bla" /label=AmpR promoter CDS 4682..5542 /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(4900..4919) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5713..6301 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6202..6221 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat 181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata 241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa 301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg 361 gctttatata tcttgtggaa aggacgaaac accggaagag cgagctcttc tgttttagag 421 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 481 tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta gtccgtagcg 541 cgtgcgccaa ttctgcagac aaattctaga ggtacccgtt acataactta cggtaaatgg 601 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg 661 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 721 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 781 ctggcattgt gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 841 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat 901 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc 961 gatgggggcg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg 1021 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc 1081 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg 1141 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc 1201 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 1261 cgggctgtaa ttagctgagc aagaggtaag ggtttaaggg atggttggtt ggtggggtat 1321 taatgtttaa ttacctggag cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc 1381 caccatgccc aagaagaaga ggaaggtgtc caatctcctg actgttcacc agaacctccc 1441 tgcgctgcca gtagatgcca ctagcgatga ggtcaggaaa aatctcatgg atatgtttag 1501 ggatagacag gcgttttctg aacacacctg gaaaatgctg cttagcgtgt gccgatcctg 1561 ggcagcctgg tgtaagctga acaatcgcaa atggttcccc gccgagccgg aggacgtgcg 1621 cgattacctg ctgtatctcc aggcaagagg gctggctgtc aagactatcc agcagcactt 1681 gggccaactg aatatgctgc atcgacgcag cgggctcccc cggcctagcg attcaaacgc 1741 agtctccctt gttatgagga gaattagaaa ggaaaacgta gatgcgggtg agagggctaa 1801 gcaggctctc gcttttgagc ggactgattt cgaccaggtc agatccctga tggagaacag 1861 cgatcggtgc caggacatca ggaacctcgc atttctggga attgcatata acacacttct 1921 gcgcatagct gagatcgccc ggatcagagt gaaagacatc agtcgaacgg acggcggccg 1981 gatgcttatt catattggac gcacaaagac attggtcagc accgctggcg ttgaaaaggc 2041 cttgtccctg ggcgtaacga agctggtgga aagatggatc tcagtgtccg gcgtggctga 2101 cgaccctaat aattacttgt tctgtcgagt gagaaaaaac ggagtcgccg cgccctctgc 2161 caccagccaa ttgagtacac gggcccttga agggatcttt gaggcaaccc accgactcat 2221 atacggagcc aaggatgaca gtggccagag gtatctcgcc tggtcaggtc attctgctag 2281 ggtgggggcc gcacgagaca tggcgcgggc aggagtctcc ataccagaga ttatgcaagc 2341 tggaggttgg acaaatgtga acatcgttat gaactatatc cgcaatcttg actctgaaac 2401 cggggccatg gtgagactgc tcgaagatgg tgactaccca tacgatgttc cagattacgc 2461 ttaagaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag 2521 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat 2581 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc 2641 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg 2701 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct 2761 ttccggaact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct 2821 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg 2881 gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac 2941 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct 3001 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct 3061 ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg gtggcatccc 3121 tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc 3181 cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt 3241 atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc 3301 tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc 3361 tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag 3421 gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata 3481 ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa 3541 ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta 3601 accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc 3661 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 3721 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt 3781 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta 3841 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 3901 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 3961 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 4021 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc 4081 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 4141 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata 4201 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa 4261 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg 4321 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg 4381 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 4441 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat 4501 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 4561 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 4621 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 4681 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 4741 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 4801 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 4861 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 4921 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 4981 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 5041 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 5101 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 5161 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 5221 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 5281 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 5341 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 5401 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 5461 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 5521 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 5581 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 5641 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 5701 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 5761 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 5821 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 5881 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 5941 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 6001 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 6061 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 6121 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 6181 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 6241 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 6301 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 6361 gt //
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