质粒名称: | pcDNA3.1 MCS-BirA(R118G)-HAP |
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出品公司: | addgene |
目录编号: | 36047 |
存储实验室: | Kyle Roux |
载体骨架基本信息 |
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载体名称: | pcDNA3.1 |
载体出品公司: | Invitrogen |
空载体大小: | 5333bp |
载体类型: | 哺乳细胞表达 |
筛选标记: | 新霉素(G418) |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | XL1 Blue |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | BirA(R118G)-HA |
物种来源: | E. coli |
插入大小: | 993bp |
基因突变: | R118G (highly promiscuous form) |
标签/融合蛋白: | HA (C terminal on insert) |
克隆方法: | 未知 |
5’测序引物 | CMV-F |
3’测序引物 | BGH-rev |
文献引用方法 |
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材料和方法部分: | pcDNA3.1 MCS-BirA(R118G)-HA was a gift from Kyle Roux (Addgene plasmid # 36047 ; http://n2t.net/addgene:36047 ; RRID:Addgene_36047) |
参考文献部分: | A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. Roux KJ, Kim DI, Raida M, Burke B. J Cell Biol. 2012 Mar 12. 10.1083/jcb.201112098 PubMed 22412018 |
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载体基本信息
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载体序列
LOCUS pcDNA3.1 MCS-BirA(R118G)-HAP 6380 bp ds-DNA circular SYN 05-JAN-2023 DEFINITION To fuse your protein of interest to the N-terminus of BirA(R118G) and use in proximity-dependent biotin identification (BioID); HA tag. ACCESSION . VERSION . KEYWORDS pcDNA3.1 MCS-BirA(R118G)-HA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6380) AUTHORS Roux KJ, Kim DI, Raida M, Burke B TITLE A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. JOURNAL J Cell Biol. 2012 Mar 12. PUBMED 22412018 REFERENCE 2 (bases 1 to 6380) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 5, 2023 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..6380 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(90..109) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 281..660 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 661..864 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 815..835 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 909..928 /label=T7 /note="T7 promoter, forward primer" promoter 909..927 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 997..1956 /codon_start=1 /product="promiscuous R118G mutant of the E. coli biotin protein ligase (Cronan, 2005)" /label=BirA(R118G) /note="also known as BirA* or BioID" /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD ACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK" CDS 1960..1986 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(2017..2034) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2023..2247 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2293..2721 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2380..2399) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2590..2611 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2730..2750) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2735..3064 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2915..3050 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2977..2996 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3131..3925 /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(3185..3204) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3795..3814 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4099..4220 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4136..4155) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4190..4209 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4269..4285) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4269..4285) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4282..4304) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4293..4309 /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(4317..4347) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4362..4383 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4500..4517) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4671..5259) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4751..4770) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5430..6290) /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 6053..6072 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6291..15) /gene="bla" /label=AmpR promoter ORIGIN 1 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga 61 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 121 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa 181 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt 241 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt 301 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 361 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 421 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 481 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 541 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 601 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 661 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 721 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 781 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 841 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt 901 atcgaaatta atacgactca ctatagggag acccaagctg gctagcgctt aaggcctgtt 961 aaccggtcgt acgtctccgg attcgaattc ggatccaagg acaacaccgt gcccctgaag 1021 ctgatcgccc tgctggccaa cggcgagttc cactctggcg agcagctggg agagaccctg 1081 ggaatgagca gagccgccat caacaagcac atccagacac tgagagactg gggagtggac 1141 gtgttcaccg tgcctggcaa gggctacagc ctgcctgagc ctatccagct gctgaacgcc 1201 aagcagatcc tgggacagct ggatggcgga agcgtggccg tgctgcctgt gatcgactcc 1261 accaatcagt acctgctgga cagaatcgga gagctgaagt ccggcgacgc ctgcatcgcc 1321 gagtaccagc aggctggcag aggaggcaga ggacggaagt ggttcagccc attcggagcc 1381 aacctgtacc tgtccatgtt ctggagactg gagcagggac ctgctgctgc catcggactg 1441 agtctggtga tcggaatcgt gatggccgag gtgctgagaa agctgggagc cgacaaggtg 1501 agagtgaagt ggcctaatga cctgtacctc caggaccgca agctggctgg catcctggtg 1561 gagctgacag gcaagacagg cgatgccgct cagatcgtga tcggagccgg aatcaacatg 1621 gccatgagaa gagtggagga gagcgtggtg aaccagggct ggatcaccct gcaggaggct 1681 ggcatcaacc tggaccggaa caccctggcc gccatgctga tcagagagct gagagccgct 1741 ctggagctgt tcgagcagga gggactggct ccttacctga gcagatggga gaagctggac 1801 aacttcatca acagacctgt gaagctgatc atcggcgaca aggaaatctt cggcatctcc 1861 agaggaatcg acaagcaggg agctctgctg ctggagcagg acggaatcat caagccctgg 1921 atgggcggag aaatctccct gagaagcgca gagaaggctt acccttacga tgtaccggat 1981 tacgcatagg cggcctaagt ttaaaccgct gatcagcctc gactgtgcct tctagttgcc 2041 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 2101 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 2161 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc 2221 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta 2281 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 2341 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 2401 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 2461 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 2521 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 2581 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 2641 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 2701 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc 2761 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag 2821 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 2881 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 2941 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 3001 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg 3061 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg 3121 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga 3181 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt 3241 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct 3301 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg 3361 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt 3421 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc 3481 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc 3541 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga 3601 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg 3661 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat 3721 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg 3781 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc 3841 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta 3901 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg 3961 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc 4021 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg 4081 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat 4141 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 4201 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg 4261 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 4321 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 4381 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 4441 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 4501 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 4561 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 4621 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 4681 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 4741 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 4801 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 4861 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 4921 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 4981 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 5041 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 5101 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 5161 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 5221 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 5281 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 5341 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 5401 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 5461 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 5521 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 5581 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 5641 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 5701 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 5761 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 5821 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 5881 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 5941 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 6001 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 6061 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 6121 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 6181 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 6241 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 6301 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 6361 tgtatttaga aaaataaaca //