出品公司: | OriGene |
---|---|
载体名称: | pCas-Guide-Nickase |
质粒类型: | CRISPR/Cas9载体;基因敲除载体;哺乳动物细胞载体 |
高拷贝/低拷贝: | 高拷贝 |
插入位点: | 限制性内切酶,多克隆位点 |
启动子: | U6 |
载体大小: | 8033 bp |
5' 测序引物及序列: | 5’ gatcgATGGGAGGTGGTATGGGAGGg 3’ |
3' 测序引物及序列: | 5’ aaaacCCTCCCATACCACCTCCCATc 3’ |
载体标签: | Myc(C-ter) |
载体抗性: | 氨苄青霉素 |
筛选标记: | -- |
克隆菌株: | Stbl3 |
宿主细胞(系): | 哺乳动物细胞 |
备注: | -- |
产品目录号: | GE100019 |
稳定性: | 瞬表达 |
组成型/诱导型: | 组成型 |
病毒/非病毒: | 非病毒 |
买家导航
pCas-Guide-Nickase载体质粒基本信息
pCas-Guide-Nickase质粒图谱载体图谱和pCas-Guide-Nickase载体序列质粒序列多克隆位点信息
pCas-Guide-Nickase质粒载体简介
pCas-Guide-Nickase载体特征
Expresses human codon-optimized Cas9 nickase D10A mutant, C-terminal Myc-DDK tagged
BamH I and BsmB I can be used for genomic target sequence cloning
Expresses gRNA containing the inserted target sequence and the gRNA scaffold
CF3 primer can be used to sequence the target sequence after cloning
pCas-Guide-Nickase质粒序列载体序列
LOCUS Exported File 8033 bp ds-DNA circular SYN 23-七月-2016 DEFINITION Mammalian vector for co-expressing a guide RNA (gRNA) together with the D10A nickase mutant of Cas9, for genome editing using the CRISPR/Cas9 system. ACCESSION . VERSION . KEYWORDS pCas-Guide-Nickase SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8033) AUTHORS OriGene TITLE Direct Submission JOURNAL Exported 2016-07-23 from SnapGene Viewer 1.5.3 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8033 /organism="synthetic DNA construct" /lab_host="Mammalian Cells" /mol_type="other DNA" promoter 90..330 /note="U6 promoter" /note="RNA polymerase III promoter for human U6 snRNA" /note="color: #000000; direction: RIGHT" misc_RNA 384..459 /note="gRNA scaffold" /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" /note="color: #00ccff; direction: RIGHT" terminator 460..465 /note="polIII terminator" /note="RNA polymerase III transcription terminator" /note="color: #ffffff" enhancer 511..890 /note="CMV enhancer" /note="human cytomegalovirus immediate early enhancer" /note="color: #000000" promoter 891..1094 /note="CMV promoter" /note="human cytomegalovirus (CMV) immediate early promoter" /note="color: #000000; direction: RIGHT" promoter 1120..1138 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" /note="color: #ffffff; direction: RIGHT" CDS 1197..5300 /codon_start=1 /locus_tag=" " /product="nickase mutant of the Cas9 endonuclease from the?Streptococcus pyogenes?Type II CRISPR/Cas system" /note="Cas9(D10A)" /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /note="This feature has 3 segments: ???1:?1197?..?1223?/?#993366 ???2:?1224?..?1226?/?#cc99b2 ???3:?1227?..?5300?/?#993366" /protein_id=" " /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 5301..5321 /codon_start=1 /locus_tag=" " /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /note=" " /note="color: #993366" /protein_id=" " /translation="PKKKRKV" CDS 5328..5348 /codon_start=1 /locus_tag=" " /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /note=" " /note="color: #993366" /protein_id=" " /translation="PKKKRKV" CDS 5370..5399 /codon_start=1 /product="Myc (human c-Myc oncogene) epitope tag" /note="Myc" /note="color: #cc99b2" /translation="EQKLISEEDL" CDS 5418..5441 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /note="FLAG" /note="color: #cc99b2" /translation="DYKDDDDK" polyA_signal 5488..6110 /note="hGH poly(A) signal" /note="human growth hormone polyadenylation signal" /note="color: #a6acb3" rep_origin complement(6231..6819) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /note="color: #ffff00" CDS complement(6994..7854) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /note="This feature has 2 segments: ???1:?6994?..?7785?/?#ccffcc ???2:?7786?..?7854?/?#ccffcc?/?signal?sequence Cleavage site after base 7785" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" ORIGIN 1 gaattcccca gtggaaagac gcgcaggcaa aacgcaccac gtgacggagc gtgaccgcgc 61 gccgagcgcg cgccaaggtc gggcaggaag agggcctatt tcccatgatt ccttcatatt 121 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa 181 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt 241 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt 301 tcttgggttt atatatcttg tggaaaggac gcgggatcca ctggaccagg cagcagcgtc 361 agaagacttt tttggaacgt ctcgttttag agctagaaat agcaagttaa aataaggcta 421 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttggtg tacatttata 481 ttggctcatg tccaatatga ccgccatgtt gacattgatt attgactagt tattaatagt 541 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 601 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 661 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 721 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta 781 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg 841 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 901 tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc 961 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat 1021 gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 1081 atataagcag agctcgttta gtgaaccgtc agaattttgt aatacgactc actatagggc 1141 ggccgggaat tcgtcgactg gaaccggtac cgaggagatc tgccgccgcg atcgccatgg 1201 ataagaaata ctcaatagga ctggctattg gcacaaatag cgtcggatgg gctgtgatca 1261 ctgatgaata taaggttcct tctaaaaagt tcaaggttct gggaaataca gaccgccaca 1321 gtatcaaaaa aaatcttata ggggctcttc tgtttgacag tggagagaca gccgaagcta 1381 ctagactcaa acggacagct aggagaaggt atacaagacg gaagaatagg atttgttatc 1441 tccaggagat tttttcaaat gagatggcca aagtggatga tagtttcttt catagacttg 1501 aagagtcttt tttggtggaa gaagacaaga agcatgaaag acatcctatt tttggaaata 1561 tagtggatga agttgcttat cacgagaaat atccaactat ctatcatctg agaaaaaaat 1621 tggtggattc tactgataaa gccgatttgc gcctgatcta tttggccctg gcccacatga 1681 ttaagtttag aggtcatttt ttgattgagg gcgatctgaa tcctgataat agtgatgtgg 1741 acaaactgtt tatccagttg gtgcaaacct acaatcaact gtttgaagaa aaccctatta 1801 acgcaagtgg agtggatgct aaagccattc tttctgcaag attgagtaaa tcaagaagac 1861 tggaaaatct cattgctcag ctccccggtg agaagaaaaa tggcctgttt gggaatctca 1921 ttgctttgtc attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg 1981 ctaaactcca gctttcaaaa gatacttacg atgatgatct ggataatctg ttggctcaaa 2041 ttggagatca atatgctgat ttgtttttgg cagctaagaa tctgtcagat gctattctgc 2101 tttcagacat cctgagagtg aatactgaaa taactaaggc tcccctgtca gcttcaatga 2161 ttaaacgcta cgatgaacat catcaagact tgactcttct gaaagccctg gttagacaac 2221 aacttccaga aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt 2281 atattgatgg cggcgcaagc caagaagaat tttataaatt tatcaaacca attctggaaa 2341 aaatggatgg tactgaggaa ctgttggtga aactgaatag agaagatttg ctgcgcaagc 2401 aacggacctt tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta 2461 ttttgagaag acaagaagac ttttatccat ttctgaaaga caatagagag aagattgaaa 2521 aaatcttgac ttttaggatt ccttattatg ttggtccatt ggccagaggc aatagtaggt 2581 ttgcatggat gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg 2641 tcgataaagg tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc 2701 ttccaaatga aaaagtgctg ccaaaacata gtttgcttta tgagtatttt accgtttata 2761 acgaattgac aaaggtcaaa tatgttactg aaggaatgag aaaaccagca tttctttcag 2821 gtgaacagaa gaaagccatt gttgatctgc tcttcaaaac aaataggaaa gtgaccgtta 2881 agcaactgaa agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag 2941 gagttgaaga tagatttaat gcttcactgg gtacatacca tgatttgctg aaaattatta 3001 aagataaaga ttttttggat aatgaagaaa atgaagacat cctggaggat attgttctga 3061 cattgaccct gtttgaagat agggagatga ttgaggaaag acttaaaaca tacgctcacc 3121 tctttgatga taaggtgatg aaacagctta aaagacgcag atatactggt tggggaaggt 3181 tgtccagaaa attgattaat ggtattaggg ataagcaatc tggcaaaaca atactggatt 3241 ttttgaaatc agatggtttt gccaatcgca attttatgca gctcatccat gatgatagtt 3301 tgacatttaa agaagacatc caaaaagcac aagtgtctgg acaaggcgat agtctgcatg 3361 aacatattgc aaatctggct ggtagccctg ctattaaaaa aggtattctc cagactgtga 3421 aagttgttga tgaattggtc aaagtgatgg ggcggcataa gccagaaaat atcgttattg 3481 aaatggcaag agaaaatcag acaactcaaa agggccagaa aaattccaga gagaggatga 3541 aaagaatcga agaaggtatc aaagaactgg gaagtcagat tcttaaagag catcctgttg 3601 aaaatactca attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagata 3661 tgtatgtgga ccaagaactg gatattaata ggctgagtga ttatgatgtc gatcacattg 3721 ttccacaaag tttccttaaa gacgattcaa tagacaataa ggtcctgacc aggtctgata 3781 aaaatagagg taaatccgat aacgttccaa gtgaagaagt ggtcaaaaag atgaaaaact 3841 attggagaca acttctgaac gccaagctga tcactcaaag gaagtttgat aatctgacca 3901 aagctgaaag aggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg 3961 ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta 4021 aatacgatga aaatgataaa cttattagag aggttaaagt gattaccctg aaatctaaac 4081 tggtttctga cttcagaaaa gatttccaat tctataaagt gagagagatt aacaattacc 4141 atcatgccca tgatgcctat ctgaatgccg tcgttggaac tgctttgatt aagaaatatc 4201 caaaacttga aagcgagttt gtctatggtg attataaagt ttatgatgtt aggaaaatga 4261 ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa gtatttcttt tactctaata 4321 tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc 4381 tgatcgaaac taatggggaa actggagaaa ttgtctggga taaagggaga gattttgcca 4441 cagtgcgcaa agtgttgtcc atgccccaag tcaatatcgt caagaaaaca gaagtgcaga 4501 caggcggatt ctctaaggag tcaattctgc caaaaagaaa ttccgacaag ctgattgcta 4561 ggaaaaaaga ctgggaccca aaaaaatatg gtggttttga tagtccaacc gtggcttatt 4621 cagtcctggt ggttgctaag gtggaaaaag ggaaatccaa gaagctgaaa tccgttaaag 4681 agctgctggg gatcacaatt atggaaagaa gttcctttga aaaaaatccc attgactttc 4741 tggaagctaa aggatataag gaagttaaaa aagacctgat cattaaactg cctaaatata 4801 gtctttttga gctggaaaac ggtaggaaac ggatgctggc tagtgccgga gaactgcaaa 4861 aaggaaatga gctggctctg ccaagcaaat atgtgaattt tctgtatctg gctagtcatt 4921 atgaaaagtt gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc 4981 ataagcatta tctggatgag attattgagc aaatcagtga attttctaag agagttattc 5041 tggcagatgc caatctggat aaagttctta gtgcatataa caaacataga gacaaaccaa 5101 taagagaaca agcagaaaat atcattcatc tgtttacctt gaccaatctt ggagcacccg 5161 ctgcttttaa atactttgat acaacaattg ataggaaaag atatacctct acaaaagaag 5221 ttctggatgc cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt 5281 tgagtcagct gggaggtgac cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc 5341 ggaaagtgga cacgcgtacg cggccgctcg agcagaaact catctcagaa gaggatctgg 5401 cagcaaatga tatcctggat tacaaggatg acgacgataa ggtttaaacg gccggccgcg 5461 gtcatagctg tttcctgaac agatcccggg tggcatccct gtgacccctc cccagtgcct 5521 ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt 5581 tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta 5641 tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca 5701 agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg 5761 attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc 5821 taatttttgt ttttttggta gaggcggggt ttcaccatat tggccaggct ggtctccaac 5881 tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga 5941 accactgctc ccttccctgt ccttctgatt ttaaaataac tataccagca ggaggacgtc 6001 cagacacagc ataggctacc tggccatgcc caaccggtgg gacatttgag ttgcttgctt 6061 ggcactgtcc tctcatgcgt tgggtccact cagtagatgc ctgttgaatt gggtacgcgg 6121 ccagcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 6181 ggataacgca ggaaagaaca tgtccgtaaa aaggccgcgt tgctggcgtt tttccatagg 6241 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 6301 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 6361 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 6421 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 6481 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 6541 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 6601 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 6661 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 6721 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 6781 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 6841 acggggtctg acgctcagtg gaacgacgcg taactcacgt taagggattt tggtcatgag 6901 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 6961 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 7021 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 7081 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 7141 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 7201 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 7261 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 7321 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 7381 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 7441 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 7501 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 7561 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 7621 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 7681 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 7741 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 7801 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 7861 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgatga tattatttta 7921 tcttgtgcaa tgtaacatca gagattttga gacacgggcc agagctgcca ggaaacagct 7981 atgaccatgt aatacgactc actatagggg atatcagctg gatggcagtt aac //
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pCas-Guide-Nickase质粒载体应用举例
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