| 质粒名称: | pcDNA3.1 MCS-BirA(R118G)-HAP |
|---|---|
| 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 |
|
| 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 |
|
| 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 |
|
| 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 |
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
-HAP质粒图谱.png)
载体简介
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
//
