| 质粒名称: | pEF6a-BCCP-PupE |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 113399 |
| 存储实验室: | Min Zhuang |
|
载体骨架基本信息 |
|
| 载体名称: | pEF6a |
| 完整质粒大小: | 6306bp |
| 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 |
|
| 插入基因名称: | BCCP-PupE |
| 物种来源: | Synthetic |
| 插入大小: | 516 bp |
| 基因突变: | T2A, S13L, A92T, V104T, V114I, E161K, Y193K, F198Y, D202T, I203V, Y258F, A283V, K288T, N290Y relative to RpBphP2 |
| 启动子: | EF-1α |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | EF-1α |
|
文献引用方法 |
|
| 材料和方法部分: | pEF6a-BCCP-PupE was a gift from Min Zhuang (Addgene plasmid # 113399 ; http://n2t.net/addgene:113399 ; RRID:Addgene_113399) |
| 参考文献部分: | A proximity-tagging system to identify membrane protein-protein interactions. Liu Q, Zheng J, Sun W, Huo Y, Zhang L, Hao P, Wang H, Zhuang M. Nat Methods. 2018 Aug 13. pii: 10.1038/s41592-018-0100-5. doi: 10.1038/s41592-018-0100-5. 10.1038/s41592-018-0100-5 PubMed 30104635 |
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 pEF6a-BCCP-PupE 6306 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses BCCP-PupE in mammalian cell.
ACCESSION .
VERSION .
KEYWORDS pEF6a-BCCP-PupE
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6306)
AUTHORS Liu Q, Zheng J, Sun W, Huo Y, Zhang L, Hao P, Wang H, Zhuang M
TITLE A proximity-tagging system to identify membrane protein-protein
interactions.
JOURNAL Nat Methods. 2018 Aug 13. pii: 10.1038/s41592-018-0100-5. doi:
10.1038/s41592-018-0100-5.
PUBMED 30104635
REFERENCE 2 (bases 1 to 6306)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6306
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..1179
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 232..1170
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 1127..1147
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
primer_bind 1196..1215
/label=T7
/note="T7 promoter, forward primer"
promoter 1196..1214
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1277..1294
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 1559..1582
/codon_start=1
/product="recognition and cleavage site for human
rhinovirus 3C and PreScission proteases"
/label=HRV 3C site
/translation="LEVLFQGP"
CDS 1838..1867
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 1883..1900
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(1923..1940)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 1929..2153
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 2199..2627
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2286..2305)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2496..2517
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2636..2656)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 2641..2970
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 2821..2956
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 2883..2902
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 3018..3065
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 3084..3482
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label=BSD
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
polyA_signal 3640..3761
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(3677..3696)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 3731..3750
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(3810..3826)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3810..3826)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(3823..3845)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 3834..3850
/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(3858..3888)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3903..3924
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4041..4058)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4201..4789)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(4281..4300)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(4960..5820)
/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 5583..5602
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(5821..5925)
/gene="bla"
/label=AmpR promoter
primer_bind complement(6000..6019)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gaattagctt ggtactaata
1201 cgactcacta tagggagacc caagctggct aggtaagctt ggtaccgagc tcggatccgc
1261 caccatgagg ggcagccacc accatcatca ccacagccag gatccggccg ggaaggcagg
1321 cgagggagag atccccgcac ccttggccgg cacggtcagc aaaatcctgg tcaaggaagg
1381 cgacaccgtg aaggctggac agacggtgtt ggtactggag gcgatgaaga tggagacaga
1441 gatcaatgcc ccgaccgatg ggaaggtgga gaaggtgttg gttaaggaga gggacgccgt
1501 gcagggcggt cagggactga tcaagatcgg cgactacgac atcccgacaa ccgccagcct
1561 ggaagttctg ttccaggggc ccggatccat gaacgcgaaa cagacccaga tcatgggtgg
1621 cggtggtcgt gacgaagaca atgcggaaga ctctgctcag gcgtctggtc aggttcagat
1681 caataccgaa ggtgttgact ctctgctgga cgaaatcgac ggcctgctcg aaaacaacgc
1741 ggaggaattc gttcgttctt acgttcagaa aggtggtgaa taagaattct gcagatatcc
1801 agcacagtgg cggccgctcg agtctagagg gcccttcgaa caaaaactca tctcagaaga
1861 ggatctgaat atgcataccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc
1921 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
1981 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
2041 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
2101 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
2161 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
2221 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
2281 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
2341 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
2401 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
2461 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
2521 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
2581 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
2641 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
2701 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
2761 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
2821 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
2881 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
2941 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
3001 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat
3061 acgacaaggt gaggaactaa accatggcca agcctttgtc tcaagaagaa tccaccctca
3121 ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca
3181 gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg
3241 ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc
3301 tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt
3361 gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg
3421 gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct
3481 aagcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc
3541 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
3601 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
3661 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
3721 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
3781 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
3841 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
3901 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
3961 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
4021 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
4081 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
4141 gcaggaaaga acatgtgagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
4201 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
4261 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
4321 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
4381 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
4441 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
4501 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
4561 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
4621 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
4681 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
4741 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
4801 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
4861 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
4921 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
4981 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
5041 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
5101 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
5161 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
5221 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
5281 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
5341 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
5401 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
5461 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
5521 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
5581 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
5641 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
5701 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
5761 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
5821 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
5881 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
5941 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
6001 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
6061 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
6121 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
6181 tatacgcgtt gacattgatt attgactagg cttttgcaaa aagctttgca aagatggata
6241 aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt gaaaggagtg
6301 ggaatt
//
