| 质粒名称: | pET21a-BirA |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 20857 |
| 存储实验室: | Alice Ting |
|
载体骨架基本信息 |
|
| 载体名称: | pET21a |
| 空载体大小: | 5443bp |
| 完整质粒大小: | 6389 bp |
| 载体类型: | 细菌细胞表达 |
| 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: | BirA |
| 物种来源: | E. Coli |
| 插入大小: | 963bp |
| 标签/融合蛋白: | His (C terminal on backbone) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | EcoRI(未破坏) |
| 3’克隆位点: | HindIII (未破坏) |
| 5’测序引物 | T7 |
| 3’测序引物 | T7 term |
|
文献引用方法 |
|
| 材料和方法部分: | pET21a-BirA was a gift from Alice Ting (Addgene plasmid # 20857 ; http://n2t.net/addgene:20857 ; RRID:Addgene_20857) |
| 参考文献部分: | Targeting quantum dots to surface proteins in living cells with biotin ligase. Howarth M, Takao K, Hayashi Y, Ting AY. Proc Natl Acad Sci U S A. 2005 May 24. 102(21):7583-8. 10.1073/pnas.0503125102 PubMed 15897449 |
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 pET21a-BirA 6389 bp ds-DNA circular SYN 14-FEB-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pET21a-BirA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6389)
AUTHORS Howarth M, Takao K, Hayashi Y, Ting AY
TITLE Targeting quantum dots to surface proteins in living cells with
biotin ligase.
JOURNAL Proc Natl Acad Sci U S A. 2005 May 24. 102(21):7583-8.
PUBMED 15897449
REFERENCE 2 (bases 1 to 6389)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Feb 14, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6389
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 939..958
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(978..1055)
/gene="lacI"
/label=lacI promoter
primer_bind 1261..1280
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 1364..1383
/label=T7
/note="T7 promoter, forward primer"
promoter 1364..1382
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 1383..1407
/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)."
RBS 1422..1444
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 1452..1484
/codon_start=1
/product="leader peptide from bacteriophage T7 gene 10"
/label=T7 tag (gene 10 leader)
/note="promotes efficient translation in E. coli"
/translation="MASMTGGQQMG"
CDS 1500..2462
/codon_start=1
/gene="birA"
/product="E. coli biotin protein ligase"
/label=BirA
/translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
CDS 2484..2501
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(2554..2572)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 2568..2615
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 2652..3107
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2739..2758)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2949..2970
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 3133..3237
/gene="bla"
/label=AmpR promoter
CDS 3238..4098
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(3456..3475)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 4269..4857
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4758..4777
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5011..5028
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 5043..5182
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(5108..5127)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5268..5290
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(5284..5475)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(6284..977)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
ORIGIN
1 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
61 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
121 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
181 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
241 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
301 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
361 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
421 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
481 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
541 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
601 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
661 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
721 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
781 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
841 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
901 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
961 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
1021 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
1081 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
1141 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
1201 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
1261 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
1321 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
1381 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
1441 gagatataca tatggctagc atgactggtg gacagcaaat gggtcgcgga tccgaattca
1501 tgaaggataa caccgtgcca ctgaaattga ttgccctgtt agcgaacggt gaatttcact
1561 ctggcgagca gttgggtgaa acgctgggaa tgagccgggc ggctattaat aaacacattc
1621 agacactgcg tgactggggc gttgatgtct ttaccgttcc gggtaaagga tacagcctgc
1681 ctgagcctat ccagttactt aatgctaaac agatattggg tcagctggat ggcggtagtg
1741 tagccgtgct gcctgtgatt gactccacga atcagtacct tcttgatcgt atcggagagc
1801 ttaaatcggg cgatgcttgc attgcagaat accagcaggc tggccgtggt cgccggggtc
1861 ggaaatggtt ttcgcctttt ggcgcaaact tatatttgtc gatgttctgg cgtctggaac
1921 aaggcccggc ggcggcgatt ggtttaagtc tggttatcgg tatcgtgatg gcggaagtat
1981 tacgcaagct gggtgcagat aaagttcgtg ttaaatggcc taatgacctc tatctgcagg
2041 atcgcaagct ggcaggcatt ctggtggagc tgactggcaa aactggcgat gcggcgcaaa
2101 tagtcattgg agccgggatc aacatggcaa tgcgccgtgt tgaagagagt gtcgttaatc
2161 aggggtggat cacgctgcag gaagcgggga tcaatctcga tcgtaatacg ttggcggcca
2221 tgctaatacg tgaattacgt gctgcgttgg aactcttcga acaagaagga ttggcacctt
2281 atctgtcgcg ctgggaaaag ctggataatt ttattaatcg cccagtgaaa cttatcattg
2341 gtgataaaga aatatttggc atttcacgcg gaatagacaa acagggggct ttattacttg
2401 agcaggatgg aataataaaa ccctggatgg gcggtgaaat atccctgcgt agtgcagaaa
2461 aaaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
2521 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
2581 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
2641 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
2701 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
2761 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
2821 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
2881 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
2941 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
3001 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
3061 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
3121 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
3181 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
3241 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
3301 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
3361 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
3421 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
3481 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
3541 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
3601 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
3661 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
3721 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
3781 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
3841 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
3901 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
3961 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
4021 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
4081 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
4141 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
4201 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
4261 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
4321 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
4381 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
4441 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
4501 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
4561 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
4621 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
4681 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
4741 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
4801 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
4861 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
4921 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
4981 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
5041 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
5101 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
5161 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
5221 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
5281 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
5341 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
5401 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
5461 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
5521 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
5581 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
5641 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
5701 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
5761 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
5821 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
5881 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
5941 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
6001 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
6061 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
6121 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
6181 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
6241 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
6301 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
6361 tttgcgtatt gggcgccagg gtggttttt
//
