质粒名称: | pET-MBP-NLS-Geo_st |
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出品公司: | 爱迪基因 |
目录编号: | 87703 |
存储实验室: | Jennifer Doudna |
载体骨架基本信息 |
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载体名称: | pET |
载体抗性: | 氨苄青霉素 |
载体类型: | 细菌表达 |
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: | GeoCas9 |
物种来源: | Geobacillus stearothermophilus |
插入大小: | 3349 bp |
GenBank编号 | OAO79865.1 |
标签/融合蛋白: | 10xHis MBP TEV(插入件上的N端子) |
克隆方法: | Gibson克隆 |
5' 测序引物 | GATGAAGCCCTGAAAGACGCGCAG |
3' 测序引物 | CTA GTT ATT GCT CAG CGG T |
文献引用方法 |
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材料和方法部分: | pET-MBP-NLS-Geo_st was a gift from Jennifer Doudna (Addgene plasmid # 87703 ; http://n2t.net/addgene:87703 ; RRID:Addgene_87703) |
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 pET-MBP-NLS-Geo_st 9263 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Expression plasmid for Cas9 from Geobacillus stearothermophilus with an N-Term MBP and SV40 NLS. ACCESSION . VERSION . KEYWORDS pET-MBP-NLS-Geo_st SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9263) AUTHORS Jennifer Doudna lab TITLE GeoCas9 Plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 9263) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9263 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 44..63 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 147..166 /label=T7 /note="T7 promoter, forward primer" promoter 147..165 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 254..276 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 296..325 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" CDS 335..1435 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" primer_bind 1409..1432 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 1490..1510 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1514..1534 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(4939..4957) /label=T7 Term /note="T7 terminator, reverse primer" terminator 4953..5000 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(5366..5394) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(5421..5439) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5507..5611 /gene="bla" /label=AmpR promoter CDS 5612..6472 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(5830..5849) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6643..7231 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7132..7151 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7385..7402 /label=L4440 /note="L4440 vector, forward primer" misc_feature 7417..7556 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(7482..7501) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7642..7664 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(7658..7849) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat 61 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga 121 ggatcgagat ctcgatcccg cgaaattaat acgactcact atagggagac cacaacggtt 181 tccctctagt gccggctccg gagagctctt taattaagcg gccgccctgc aggactcgag 241 ttctagaaat aattttgttt aactttaaga aggagatata catatgaaat cttctcacca 301 tcaccatcac catcaccatc accatggttc ttctatgaaa atcgaagaag gtaaactggt 361 aatctggatt aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga 421 gaaagatacc ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc 481 acaggttgcg gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg 541 tggctacgct caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa 601 gctgtatccg tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat 661 cgctgttgaa gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac 721 ctgggaagag atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat 781 gttcaacctg caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc 841 gttcaagtat gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc 901 gaaagcgggt ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac 961 cgattactcc atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg 1021 cccgtgggca tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc 1081 gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc 1141 cgccagtccg aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga 1201 aggtctggaa gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga 1261 ggaagagttg gcgaaagatc cacgtattgc cgccactatg gaaaacgccc agaaaggtga 1321 aatcatgccg aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat 1381 caacgccgcc agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactaattc 1441 gagctcgaac aacaacaaca ataacaataa caacaacctc gggatcgagg aaaacctgta 1501 cttccaatcc atgcccaaga agaagaggaa ggtgggcggt tcgaatgcaa tgcgttataa 1561 gattggcctg gacatcggta ttacctctgt tggttgggca gtcatgaacc tggatatccc 1621 tcgtatcgaa gatctgggcg tgcgcatttt tgaccgtgcg gagaacccgc agaccggtga 1681 atctctggct ctgccgcgtc gtctggcacg tagcgcacgc cgccgcctgc gtcgtcgtaa 1741 acaccgtctg gagcgtattc gtcgcctggt tattcgtgaa ggcatcctga cgaaagaaga 1801 actggataaa ctgttcgaag aaaaacacga gatcgacgta tggcagctgc gtgtagaagc 1861 cctggaccgt aagctgaaca acgacgaact ggcgcgtgtc ctgctgcatc tggcaaagcg 1921 tcgtggcttc aaatctaacc gtaaatctga acgctccaat aaagagaact ccactatgct 1981 gaaacatatt gaggagaacc gtgcaattct gtctagctac cgtaccgtgg gcgaaatgat 2041 tgttaaagac ccgaaattcg cactgcataa gcgtaacaaa ggcgaaaact acaccaacac 2101 cattgcacgc gatgacctgg aacgtgaaat ccgtctgatt ttctccaaac agcgcgaatt 2161 cggcaacatg tcttgcaccg aagaattcga aaacgaatat attaccattt gggcatctca 2221 gcgtccggtg gcgtctaaag atgatatcga aaaaaaagta ggcttttgta ctttcgaacc 2281 gaaggaaaaa cgtgcgccga aagccaccta taccttccag tcttttatcg cgtgggaaca 2341 tatcaacaaa ctgcgtctga tttctccgtc tggcgcccgc ggcctgaccg acgaagaacg 2401 tcgtctgctg tatgaacaag cattccagaa aaacaaaatt acctaccacg atattcgtac 2461 cctgctgcat ctgccggacg acacctactt caagggcatc gtttacgatc gcggtgaatc 2521 tcgtaagcag aacgaaaaca ttcgtttcct ggaactggat gcataccacc agatccgtaa 2581 agctgtagat aaagtttacg gcaagggtaa atccagcagc ttcctgccga tcgactttga 2641 taccttcggt tacgcgctga ccctgtttaa agacgatgcg gatatccact cttacctgcg 2701 caacgagtac gaacagaacg gcaaacgtat gcctaacctg gctaacaaag tttacgataa 2761 cgagctgatt gaagaactgc tgaacctgtc cttcactaaa ttcggtcacc tgtctctgaa 2821 agctctgcgt tccatcctgc cgtatatgga acagggtgaa gtctactcct ccgcttgtga 2881 acgtgcaggc tacaccttca ccggtccgaa aaagaagcaa aaaactatgc tgctgccgaa 2941 catcccgccg attgcgaacc ctgtagtaat gcgtgcactg acccaggcgc gcaaagtagt 3001 caacgcgatc atcaaaaagt acggcagccc ggtttccatc catatcgaac tggcgcgcga 3061 cctgagccag acttttgacg agcgtcgtaa aactaaaaag gaacaggatg aaaaccgtaa 3121 aaaaaacgaa accgcgatcc gccagctgat ggaatacggt ctgactctga accctactgg 3181 tcacgatatt gtgaagttca agctgtggtc tgaacagaac ggtcgctgtg cttactctct 3241 gcagccgatc gagatcgaac gtctgctgga gccaggttac gttgaagtag atcatgtgat 3301 cccgtactcc cgctctctgg atgattctta taccaacaaa gttctggttc tgactcgcga 3361 aaaccgtgag aaaggcaacc gcatcccagc tgaatatctg ggtgttggca ctgagcgttg 3421 gcaacagttc gaaaccttcg tcctgaccaa taaacagttc tctaaaaaga aacgtgaccg 3481 tctgctgcgt ctgcactacg atgaaaacga agagactgaa ttcaaaaacc gtaacctgaa 3541 cgatactcgc tacatcagcc gcttcttcgc aaacttcatt cgtgaacacc tgaaatttgc 3601 ggaatccgac gataaacaga aagtttatac cgtaaacggc cgtgttaccg cccacctgcg 3661 ttctcgctgg gagttcaaca agaaccgtga ggaaagcgat ctgcaccacg ctgttgacgc 3721 cgttattgtg gcgtgcacca ccccaagcga tatcgctaag gtgaccgcat tctaccagcg 3781 tcgtgagcag aacaaggaac tggccaaaaa aaccgaaccg cattttccgc agccgtggcc 3841 gcacttcgcg gacgaactgc gtgctcgtct gtccaaacat cctaaagaaa gcatcaaagc 3901 tctgaacctg ggtaactacg atgaccaaaa actggaatct ctgcagccgg tgtttgtcag 3961 ccgtatgccg aaacgttctg ttactggcgc tgcgcaccag gaaacgctgc gccgttacgt 4021 gggcatcgac gaacgctccg gtaaaatcca gaccgtagta aaaaccaaac tgtccgagat 4081 taaactggat gcatccggcc acttcccaat gtacggtaaa gaatccgatc cacgcactta 4141 tgaagccatc cgccagcgtc tgctggagca taacaacgac ccgaaaaagg cattccagga 4201 gcctctgtac aaaccgaaaa aaaacggcga accgggcccg gtaatccgta ctgtaaaaat 4261 tatcgacacg aaaaaccagg tgatccctct gaacgatggt aaaaccgtgg cctacaattc 4321 caacatcgtt cgcgtggacg tgttcgaaaa agatggtaaa tactactgtg taccggtgta 4381 taccatggac atcatgaaag gcattctgcc gaacaaagcg attgaaccga acaagccgta 4441 ctctgaatgg aaagaaatga ccgaagatta cacgtttcgt ttcagcctgt atccgaacga 4501 cctgatccgc atcgaactgc cgcgtgaaaa aaccgttaaa accgctgcag gcgaagaaat 4561 taacgtgaaa gacgtgttcg tttactataa aacgatcgac tccgcaaacg gcggcctgga 4621 actgatttct cacgaccacc gtttctctct gcgtggcgtt ggctctcgca ccctgaaacg 4681 tttcgagaaa tatcaagttg atgttctggg taacatctat aaagtgcgtg gcgagaaacg 4741 tgtcggtctg gcgtcctccg cacacagcaa acctggcaaa accattcgtc cactgcaatc 4801 tactcgtgac taataacatt ggaagtggat aacggatccg cgatcgcggc gcgccacctg 4861 gtggccggcc ggtaccacgc gtgcgcgctg atccggctgc taacaaagcc cgaaaggaag 4921 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac 4981 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatcca caggacgggt 5041 gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag caggactggg 5101 cggcggccaa agcggtcgga cagtgctccg agaacgggtg cgcatagaaa ttgcatcaac 5161 gcatatagcg ctagcagcac gccatagtga ctggcgatgc tgtcggaatg gacgatatcc 5221 cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg 5281 gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag 5341 caatttaact gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga 5401 gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat 5461 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat 5521 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 5581 aatgcttcaa taacattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 5641 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 5701 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 5761 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 5821 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg 5881 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 5941 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 6001 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 6061 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 6121 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa 6181 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 6241 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 6301 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 6361 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 6421 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 6481 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 6541 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 6601 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 6661 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 6721 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 6781 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 6841 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 6901 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 6961 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 7021 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 7081 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 7141 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 7201 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 7261 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 7321 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 7381 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac 7441 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc 7501 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 7561 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 7621 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 7681 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 7741 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 7801 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 7861 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 7921 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 7981 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 8041 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 8101 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 8161 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 8221 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 8281 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga 8341 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg 8401 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc 8461 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg 8521 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 8581 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt 8641 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 8701 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 8761 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 8821 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 8881 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 8941 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 9001 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 9061 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc 9121 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc 9181 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc 9241 tgccaccata cccacgccga aac //
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