| 质粒名称: | sortase A pentamutant (eSrtA) in pET29 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 75144 |
| 存储实验室: | David Liu |
|
载体骨架基本信息 |
|
| 载体名称: | pET29 |
| 载体出品公司: | Novagen |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | 5370 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | sortase A pentamutant,5M SrtA,evolved sortase A (eSrtA) |
| 物种来源: | Staphylococcus aureus |
| 插入大小: | 441 bp |
| 基因突变: | P94R, D160N, D165A, K190E, K196T. Deleted amino acids 1-59 (which do not affect catalytic activity). |
| GenBank编号 | -- |
| 启动子 | T7 |
| 标签/融合蛋白: | 6x His（主干上的C端子） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | NdeI(未破坏) |
| 3' 克隆位点: | XhoI (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: | sortase A pentamutant (eSrtA) in pET29 was a gift from David Liu (Addgene plasmid # 75144 ; http://n2t.net/addgene:75144 ; RRID:Addgene_75144) |
| 参考文献部分: | A general strategy for the evolution of bond-forming enzymes using yeast display. Chen I, Dorr BM, Liu DR. Proc Natl Acad Sci U S A. 2011 Jul 12;108(28):11399-404. doi: 10.1073/pnas.1101046108. Epub 2011 Jun 22. 10.1073/pnas.1101046108 PubMed 21697512 |
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
 in pET29质粒图谱.png)
载体简介
载体序列
LOCUS sortase A pentamutant (eSrtA) in pET29 5678 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION evolved sortase A (eSrtA) pentamutant with improved kinetics and
activity.
ACCESSION .
VERSION .
KEYWORDS sortase A pentamutant (eSrtA) in pET29
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5678)
AUTHORS Chen I, Dorr BM, Liu DR
TITLE A general strategy for the evolution of bond-forming enzymes using
yeast display.
JOURNAL Proc Natl Acad Sci U S A. 2011 Jul 12;108(28):11399-404. doi:
10.1073/pnas.1101046108. Epub 2011 Jun 22.
PUBMED 21697512
REFERENCE 2 (bases 1 to 5678)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..5678
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 12..467
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(99..118)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 309..330
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(559..1374)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind 1282..1301
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 1366..1385
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 1496..2084
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1985..2004
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 2238..2255
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 2270..2409
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(2335..2354)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2495..2517
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(2511..2702)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(3511..4593)
/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"
primer_bind 4555..4574
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(4594..4671)
/gene="lacI"
/label=lacI promoter
primer_bind 4877..4896
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 4984..5003
/label=T7
/note="T7 promoter, forward primer"
promoter 4984..5002
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 5003..5027
/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 5042..5064
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 5522..5539
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(5592..5610)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 5606..5653
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
ORIGIN
1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
841 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
1021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
1081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
1141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
1201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
1261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
1321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
1381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
1441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
1501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
1561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
1621 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
1681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
1741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
1801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
1861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
1921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
1981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
2041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
2101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
2161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
2221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
2281 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
2341 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
2401 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
2461 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
2521 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
2581 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
2641 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
2701 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
2761 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
2821 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
2881 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
2941 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
3001 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
3061 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
3121 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
3181 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
3241 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
3301 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
3361 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
3421 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
3481 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
3541 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
3601 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
3661 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
3721 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
3781 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
3841 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
3901 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
3961 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
4021 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
4081 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
4141 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
4201 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
4261 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
4321 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
4381 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
4441 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
4501 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
4561 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
4621 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
4681 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
4741 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
4801 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
4861 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
4921 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga
4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
5041 ttttgtttaa ctttaagaag gagatataca tatgcaagct aaacctcaaa ttccgaaaga
5101 taaatcaaaa gtggcaggct atattgaaat tccagatgct gatattaaag aaccagtata
5161 tccaggacca gcaacacgcg aacaattaaa tagaggtgta agctttgcag aagaaaatga
5221 atcactagat gatcaaaata tttcaattgc aggacacact ttcattgacc gtccgaacta
5281 tcaatttaca aatcttaaag cagccaaaaa aggtagtatg gtgtacttta aagttggtaa
5341 tgaaacacgt aagtataaaa tgacaagtat aagaaacgtt aagccaacag ctgtagaagt
5401 tctagatgaa caaaaaggta aagataaaca attaacatta attacttgtg atgattacaa
5461 tgaagagaca ggcgtttggg aaacacgtaa aatctttgta gctacagaag tcaaactcga
5521 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
5581 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
5641 gaggggtttt ttgctgaaag gaggaactat atccggat
//
