质粒名称: | phU6-gRNA |
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出品公司: | 爱迪基因 |
目录编号: | 53188 |
存储实验室: | Charles Gersbach |
载体骨架基本信息 |
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载体名称: | pzDonor |
载体抗性: | 卡那霉素 |
空载体大小: | 3515 bp |
载体类型: | 哺乳细胞表达,CRISPR |
高拷贝/低拷贝: | 未知 |
生长条件: | 37 ℃ |
插入基因信息 |
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启动子 | human U6 |
克隆方法: | 限制性内切酶 |
3' 测序引物 | M13 Reverse |
文献引用方法 |
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材料和方法部分: | phU6-gRNA was a gift from Charles Gersbach (爱迪基因 plasmid # 53188) |
参考文献部分: | Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA. Nucleic Acids Res. 2014 Aug 13. pii: gku749. 10.1093/nar/gku749 PubMed 25122746 |
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载体基本信息
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载体简介
载体序列
LOCUS phU6-gRNA 3515 bp ds-DNA circular SYN 10-JAN-2018 DEFINITION Expresses the S. pyogenes sgRNA from the human U6 promoter. ACCESSION . VERSION . KEYWORDS phU6-gRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3515) AUTHORS Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA TITLE Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. JOURNAL Nucleic Acids Res. 2014 Aug 13. pii: gku749. PUBMED 25122746 COMMENT This file is created by Vector NTI http://www.biofeng.com/ COMMENT ORIGDB|GenBank COMMENT VNTAUTHORNAME|biofeng.com| COMMENT VNTNAME|phU6-gRNA| FEATURES Location/Qualifiers source 1..3515 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(3..458) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(140..161) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 352..371 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 585..607 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 599..616 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 600..616 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 626..645 /label=T7 /note="T7 promoter, forward primer" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 696..936 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 696..716 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 867..886 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 963..1038 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(1094..1114) /label=T3 /note="T3 promoter, forward primer" promoter complement(1094..1112) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(1133..1149) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1133..1149) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1146..1168) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 1157..1173 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(1181..1211) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1226..1247 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(1364..1381) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1535..2123) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1615..1634) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2200..2994) /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(2311..2330) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 2921..2940 /label=Neo-R /note="Neomycin resistance gene, reverse primer" promoter complement(3029..3386) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3043..3178 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(3097..3116) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind 3343..3363 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter complement(3388..3492) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtacccg 661 tctcacaggc ggatcgatcc aaggtcgggc aggaagaggg cctatttccc atgattcctt 721 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa 781 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg 841 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt 901 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggtct tcgagaagac 961 ctgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa 1021 gtggcaccga gtcggtgctt tttttctccg ctgagcgtac tgagacgccg cggtggagct 1081 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc 1141 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 1201 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 1261 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 1321 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 1381 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 1441 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 1501 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 1561 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 1621 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 1681 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 1741 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 1801 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 1861 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 1921 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 1981 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 2041 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 2101 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 2161 agtggaacga aaactcacgt taagggattt tggtcatgat cagaagaact cgtcaagaag 2221 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg 2281 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg 2341 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc 2401 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg 2461 catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc 2521 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg 2581 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc 2641 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc 2701 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc 2761 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc 2821 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag 2881 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag 2941 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa 3001 cgatcctcat cctgtctctt gatcgatctt tgcaaaagcc taggcctcca aaaaagcctc 3061 ctcactactt ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa 3121 aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg 3181 gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc 3241 tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc 3301 tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat 3361 tccacagctg gttctttccg cctcaggact cttccttttt caatattatt gaagcattta 3421 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 3481 aggggttccg cgcacatttc cccgaaaagt gccac 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