质粒名称: | miniTurbo-His6_pET21a |
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出品公司: | addgene |
目录编号: | 107172 |
存储实验室: | Alice Ting |
载体骨架基本信息 |
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载体名称: | pCDNA3 |
完整质粒大小: | 6338 bp |
载体类型: | 哺乳细胞表达 |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | miniTurbo (BirA mutant) |
基因突变: | aa1-63 deleted; Q65P, I87V, R118S, E140K, Q141R, S150G, L151P, V160A, T192A, K194I, M209V, I305 |
克隆方法: | 限制性内切酶 |
5’克隆位点: | 未知 |
3’克隆位点: | 未知 |
文献引用方法 |
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材料和方法部分: | 3xHA-miniTurbo-NLS_pCDNA3 was a gift from Alice Ting (Addgene plasmid # 107172 ; http://n2t.net/addgene:107172 ; RRID:Addgene_107172) |
参考文献部分: | Efficient proximity labeling in living cells and organisms with TurboID. Branon TC, Bosch JA, Sanchez AD, Udeshi ND, Svinkina T, Carr SA, Feldman JL, Perrimon N, Ting AY. Nat Biotechnol. 2018 Aug 20. pii: nbt.4201. doi: 10.1038/nbt.4201. 10.1038/nbt.4201 PubMed 30125270 |
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3xHA-miniTurbo-NLS_pCDNA3载体质粒基本信息
3xHA-miniTurbo-NLS_pCDNA3质粒图谱载体图谱和3xHA-miniTurbo-NLS_pCDNA3载体序列质粒序列多克隆位点信息
3xHA-miniTurbo-NLS_pCDNA3质粒载体简介
3xHA-miniTurbo-NLS_pCDNA3质粒序列载体序列
LOCUS 3xHA-miniTurbo-NLS_pCDNA3 6338 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION expresses 3xHA-tagged miniTurbo in the mammalian nucleus. ACCESSION . VERSION . KEYWORDS 3xHA-miniTurbo-NLS_pCDNA3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6338) AUTHORS Branon TC, Bosch JA, Sanchez AD, Udeshi ND, Svinkina T, Carr SA, Feldman JL, Perrimon N, Ting AY TITLE Efficient proximity labeling in living cells and organisms with TurboID. JOURNAL Nat Biotechnol. 2018 Aug 20. pii: nbt.4201. doi: 10.1038/nbt.4201. PUBMED 30125270 REFERENCE 2 (bases 1 to 6338) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..6338 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 80..101 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(220..240) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 225..554 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 405..540 /label=SV40 ori /note="SV40 origin of replication" primer_bind 467..486 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 621..1415 /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 GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(675..694) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 1285..1304 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 1589..1710 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1626..1645) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1680..1699 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(1759..1775) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1759..1775) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1772..1794) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 1783..1799 /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(1807..1837) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1852..1873 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(1990..2007) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2161..2749) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2241..2260) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2920..3780) /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 3543..3562 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3781..3885) /gene="bla" /label=AmpR promoter primer_bind complement(3960..3979) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 4151..4530 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 4531..4734 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 4685..4705 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 4779..4798 /label=T7 /note="T7 promoter, forward primer" promoter 4779..4797 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 4826..4852 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 4856..4882 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 4886..4912 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 4919..5689 /codon_start=1 /product="N-terminally truncated mutant of the E. coli biotin ligase BioID engineered for improved catalytic efficiency (Branon et al., 2018)" /label=miniTurbo /translation="IPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACI AEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADK VRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQ EAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIF GISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK" CDS 5708..5728 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5732..5752 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5756..5776 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" promoter complement(5807..5825) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(5808..5825) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(5845..5862) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5851..6075 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 6121..211 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6208..6227) /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 61 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 121 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 181 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt 241 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt 301 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc 361 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc 421 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg 481 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc 541 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga 601 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg 661 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg 721 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt 781 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg 841 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat 901 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat 961 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg 1021 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg 1081 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc 1141 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc 1201 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg 1261 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg 1321 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca 1381 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac 1441 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga 1501 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga 1561 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa 1621 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 1681 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta 1741 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 1801 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 1861 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 1921 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 1981 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 2041 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 2101 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 2161 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 2221 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 2281 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 2341 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 2401 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 2461 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 2521 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 2581 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 2641 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 2701 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 2761 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 2821 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 2881 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 2941 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 3001 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 3061 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 3121 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 3181 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 3241 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 3301 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 3361 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 3421 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 3481 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 3541 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 3601 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 3661 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 3721 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 3781 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 3841 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 3901 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt 3961 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag 4021 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat 4081 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga 4141 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta 4201 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc 4261 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 4321 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg 4381 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa 4441 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac 4501 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg 4561 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg 4621 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca 4681 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg 4741 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 4801 acccaagctt gcggccgcca ccatgtaccc gtatgatgtt ccggattacg ctggctatcc 4861 ctacgacgtg cccgactatg ccgggtaccc ctatgacgtc ccagactacg cagctagcat 4921 cccgctgctg aacgctaaac agattctggg acagctggac ggcgggagcg tggcagtcct 4981 gcctgtggtc gactccacca atcagtacct gctggatcga atcggcgagc tgaagagtgg 5041 ggatgcttgc attgcagaat atcagcaggc agggagagga agcagaggga ggaaatggtt 5101 ctctcctttt ggagctaacc tgtacctgag tatgttttgg cgcctgaagc ggggaccagc 5161 agcaatcggc ctgggcccgg tcatcggaat tgtcatggca gaagcgctgc gaaagctggg 5221 agcagacaag gtgcgagtca aatggcccaa tgacctgtat ctgcaggata gaaagctggc 5281 aggcatcctg gtggagctgg ccggaataac aggcgatgct gcacagatcg tcattggcgc 5341 cgggattaac gtggctatga ggcgcgtgga ggaaagcgtg gtcaatcagg gctggatcac 5401 actgcaggaa gcagggatta acctggacag gaatactctg gccgctatgc tgatccgaga 5461 gctgcgggca gccctggaac tgttcgagca ggaaggcctg gctccatatc tgtcacggtg 5521 ggagaagctg gataacttca tcaatagacc cgtgaagctg atcattgggg acaaagagat 5581 tttcgggatt agccggggga ttgataaaca gggagccctg ctgctggaac aggacggagt 5641 tatcaaaccc tggatgggcg gagaaatcag tctgcggtct gccgaaaagg aattcagcag 5701 ggccgacccc aagaagaaga ggaaggtgga ccccaagaag aagaggaagg tggaccccaa 5761 gaagaagagg aaggtgtgac tcgagcatgc atctagaggg ccctattcta tagtgtcacc 5821 taaatgctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 5881 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 5941 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 6001 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 6061 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc 6121 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 6181 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 6241 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 6301 gtgctttacg gcacctcgac cccaaaaaac ttgattag //