| 质粒名称: | MCS-13X Linker-BioID2-HA |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 80899 |
| 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: | Thermo Fisher Scientific |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 6335 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 高拷贝/低拷贝: | -- |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | -- |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | CMV |
| 标签/融合蛋白: |
具有 R40G 的人源化 A.aeolicus BioID2（骨架上的 C 末端）
HA（主干上的C终端）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 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: | MCS-13X Linker-BioID2-HA was a gift from Kyle Roux (Addgene plasmid # 80899 ; http://n2t.net/addgene:80899 ; RRID:Addgene_80899) |
| 参考文献部分: | An improved smaller biotin ligase for BioID proximity labeling. Kim DI, Jensen SC, Noble KA, Kc B, Roux KH, Motamedchaboki K, Roux KJ. Mol Biol Cell. 2016 Feb 24. pii: mbc.E15-12-0844. 10.1091/mbc.E15-12-0844 PubMed 26912792 |
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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 MCS-13X Linker-BioID2-HA 6335 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION To fuse your protein of interest to the N-terminus of BioID2 and use
in proximity-dependent biotin identification (BioID); MCS and a
flexible 25nm GS linker are present upstream of BioID2 and HA tags..
ACCESSION .
VERSION .
KEYWORDS MCS-13X Linker-BioID2-HA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6335)
AUTHORS Kim DI, Jensen SC, Noble KA, Kc B, Roux KH, Motamedchaboki K, Roux
KJ
TITLE An improved smaller biotin ligase for BioID proximity labeling.
JOURNAL Mol Biol Cell. 2016 Feb 24. pii: mbc.E15-12-0844.
PUBMED 26912792
REFERENCE 2 (bases 1 to 6335)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6335
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(44..63)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 235..614
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 769..789
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 863..882
/label=T7
/note="T7 promoter, forward primer"
promoter 863..881
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1182..1877
/codon_start=1
/product="promiscuous R40G mutant of a biotin protein
ligase from Aquifex aeolicus (Kim et al., 2016)"
/label=BioID2
/translation="FKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKWL
SQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKVS
GVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISEN
LKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKEI
LSGEFSLRRS"
CDS 1878..1904
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
primer_bind complement(1926..1943)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 1932..2156
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 2202..2630
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2289..2308)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2499..2520
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2639..2659)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 2644..2973
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 2824..2959
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 2886..2905
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3040..3834
/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(3094..3113)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 3704..3723
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 4008..4129
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4045..4064)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 4099..4118
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(4178..4194)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4178..4194)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(4191..4213)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 4202..4218
/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(4226..4256)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4271..4292
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4409..4426)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4580..5168)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(4660..4679)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(5339..6199)
/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 5962..5981
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(6200..6304)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
901 gcttaaggcc tgttaaccgg tcgtacgtct ccggattcga attcggatcc catctttttt
961 cggaattcgg atccggtgga ggcgggtctg gaggcggggg tagtggcggg ggtggaagcg
1021 ggggtggagg cgggtcgggt ggcggaggta gcggaggcgg tggaagtgga ggcggtggaa
1081 gtggtggagg aggttctggt ggcggaggta gcggtggcgg aggtagcgga ggcggtggaa
1141 gtggtggcgg aggtagcgga ggcggtggat cggcgcgccg tttcaagaac ctgatctggc
1201 tgaaggaggt ggacagcacc caggagagac tgaaggagtg gaacgttagc tacggcaccg
1261 ccctggtggc cgacagacag accaagggca gaggcggcct gggcagaaag tggctgagcc
1321 aggagggcgg cctgtacttc agcttcctgc tgaaccccaa ggagttcgag aacctgctgc
1381 agctgcccct ggtgctgggc ctgagcgtga gcgaggccct ggaggagatc accgagatcc
1441 ccttcagcct gaagtggccc aacgacgtgt acttccagga gaagaaggtg agcggcgtgc
1501 tgtgcgagct gagcaaggac aagctgatcg tgggcatcgg catcaacgtg aaccagagag
1561 agatccccga ggagatcaag gacagagcca ccaccctgta cgagatcacc ggcaaggact
1621 gggacagaaa ggaggtgctg ctgaaggtgc tgaagagaat cagcgagaac ctgaagaagt
1681 tcaaggagaa gagcttcaag gagttcaagg gcaagatcga gagcaagatg ctgtacctgg
1741 gcgaggaggt gaagctgctg ggcgagggca agatcaccgg caagctggtg ggcctgagcg
1801 agaagggcgg cgccctgatc ctgaccgagg agggcatcaa ggagatcctg agcggcgagt
1861 tcagcctgag aagaagctac ccttacgatg taccggatta cgcataggtt taaaccgctg
1921 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
1981 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
2041 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
2101 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
2161 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
2221 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
2281 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
2341 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
2401 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
2461 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
2521 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
2581 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
2641 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
2701 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
2761 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
2821 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
2881 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
2941 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
3001 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
3061 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
3121 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
3181 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
3241 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
3301 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
3361 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
3421 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
3481 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
3541 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
3601 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
3661 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
3721 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
3781 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
3841 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
3901 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
3961 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
4021 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
4081 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
4141 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
4201 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
4261 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
4321 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
4381 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
4441 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
4501 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
4561 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
4621 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
4681 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
4741 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
4801 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
4861 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
4921 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
4981 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
5041 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
5101 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
5161 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
5221 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
5281 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
5341 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
5401 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
5461 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
5521 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
5581 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
5641 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
5701 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
5761 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
5821 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
5881 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
5941 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
6001 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
6061 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
6121 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
6181 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
6241 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
6301 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
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