| 质粒名称: | pGS-21a his6-GST-WTPhi29DNAP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 166863 |
| 存储实验室: | Paul Blainey, Stephen Quake |
|
载体骨架基本信息 |
|
| 载体名称: | pGS-21a |
| 载体出品公司: | GenScript |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | -- |
|
插入基因信息 |
|
| 插入基因名称: | Phi29 Polymerase codon-optimized for overexpression in E. coli |
| 物种来源: | Synthetic |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | -- |
| 标签/融合蛋白: |
GST（插入件上的N端子）
his6（插件上的C端子）
|
| 克隆方法: | -- |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | -- |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pGS-21a his6-GST-WTPhi29DNAP was a gift from Paul Blainey & Stephen Quake (Addgene plasmid # 166863 ; http://n2t.net/addgene:166863 ; RRID:Addgene_166863) |
| 参考文献部分: | Digital MDA for enumeration of total nucleic acid contamination. Blainey PC, Quake SR. Nucleic Acids Res. 2011 Mar;39(4):e19. doi: 10.1093/nar/gkq1074. Epub 2010 Nov 11. 10.1093/nar/gkq1074 PubMed 21071419 |
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 pGS-21a his6-GST-WTPhi29DNAP 7824 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION To produce a dual affinity-tagged Phi29 DNA polymerase.
ACCESSION .
VERSION .
KEYWORDS pGS-21a his6-GST-WTPhi29DNAP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7824)
AUTHORS Blainey PC, Quake SR
TITLE Digital MDA for enumeration of total nucleic acid contamination.
JOURNAL Nucleic Acids Res. 2011 Mar;39(4):e19. doi: 10.1093/nar/gkq1074.
Epub 2010 Nov 11.
PUBMED 21071419
REFERENCE 2 (bases 1 to 7824)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7824
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 1..20
/label=T7
/note="T7 promoter, forward primer"
promoter 1..19
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 20..44
/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 59..81
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 89..742
/codon_start=1
/product="glutathione S-transferase from Schistosoma
japonicum"
/label=GST
/translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
primer_bind 700..722
/label=pGEX 5'
/note="pGEX vectors, Glutathione-S-transferase, forward
primer"
CDS 779..793
/codon_start=1
/product="enterokinase recognition and cleavage site"
/label=enterokinase site
/translation="DDDDK"
CDS 2552..2569
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(2622..2640)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 2636..2683
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 2720..3175
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2807..2826)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3017..3038
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 3201..3305
/gene="bla"
/label=AmpR promoter
CDS 3306..4166
/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(3524..3543)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 4337..4925
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4826..4845
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5079..5096
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 5111..5250
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(5176..5195)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5336..5358
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(5352..5543)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(6352..7434)
/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 7396..7415
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(7435..7512)
/gene="lacI"
/label=lacI promoter
primer_bind 7718..7737
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
ORIGIN
1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
61 tgtttaactt taagaaggag atatacatat gtcccctata ctaggttatt ggaaaattaa
121 gggccttgtg caacccactc gacttctttt ggaatatctt gaagaaaaat atgaagagca
181 tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga
241 gtttcccaat cttccttatt atattgatgg tgatgttaaa ttaacacagt ctatggccat
301 catacgttat atagctgaca agcacaacat gttgggtggt tgtccaaaag agcgtgcaga
361 gatttcaatg cttgaaggag cggttttgga tattagatac ggtgtttcga gaattgcata
421 tagtaaagac tttgaaactc tcaaagttga ttttcttagc aagctacctg aaatgctgaa
481 aatgttcgaa gatcgtttat gtcataaaac atatttaaat ggtgatcatg taacccatcc
541 tgacttcatg ttgtatgacg ctcttgatgt tgttttatac atggacccaa tgtgcctgga
601 tgcgttccca aaattagttt gttttaaaaa acgtattgaa gctatcccac aaattgataa
661 gtacttgaaa tccagcaagt atatagcatg gcctttgcag ggctggcaag ccacgtttgg
721 tggtggcgac catcctccaa aatcggatct gggccacaca ggccatagat ctggtaccga
781 cgacgacgac aaggccatgg ctgatatcgg atccatgccg cgtaaaatgt attcctgtga
841 ttttgaaacg acgacgaaag ttgaagattg ccgtgtgtgg gcgtatggtt atatgaacat
901 cgaagaccat tcagaatata aaattggcaa ctcgctggat gaatttatgg cgtgggtgct
961 gaaagttcag gccgacctgt acttccacaa tctgaaattt gatggtgcgt tcattatcaa
1021 ctggctggaa cgtaatggct ttaaatggag cgccgatggt ctgccgaaca cctataatac
1081 gattatctct cgtatgggcc aatggtatat gattgatatc tgcctgggct acaaaggtaa
1141 acgcaaaatt cataccgtga tctatgacag cctgaaaaaa ctgccgtttc cggtgaagaa
1201 aattgcgaaa gatttcaaac tgaccgtcct gaaaggcgat attgactatc acaaagaacg
1261 tccggttggt tacaaaatca cgccggaaga atatgcgtac attaaaaacg atatccagat
1321 tatcgcagaa gctctgctga ttcagtttaa acaaggcctg gatcgcatga ccgccggcag
1381 tgactccctg aaaggtttca aagatatcat caccacgaaa aaattcaaaa aagtgttccc
1441 gaccctgagc ctgggtctgg ataaagaagt tcgttatgca taccgcggcg gttttacgtg
1501 gctgaacgac cgtttcaaag aaaaagaaat tggcgagggt atggtctttg atgtgaatag
1561 tctgtatccg gctcagatgt actcccgcct gctgccgtat ggcgaaccga tcgttttcga
1621 gggtaaatat gtctgggatg aagactaccc gctgcatatt cagcacatcc gttgtgaatt
1681 tgaactgaaa gaaggctata ttccgaccat tcaaatcaaa cgtagccgct tctataaggg
1741 taacgaatac ctgaaaagct ctggcggtga aatcgcagac ctgtggctga gtaacgtcga
1801 tctggaactg atgaaagaac attacgatct gtacaacgtt gaatacatct ccggcctgaa
1861 atttaaagcc accacgggtc tgtttaaaga tttcattgac aaatggacct acatcaaaac
1921 cacgtctgaa ggtgcaatca aacagctggc taaactgatg ctgaacagcc tgtatggcaa
1981 atttgcatct aatccggatg ttaccggtaa agtcccgtac ctgaaagaaa atggcgctct
2041 gggttttcgc ctgggcgaag aagaaaccaa agatccggtg tatacgccga tgggtgtttt
2101 cattaccgcg tgggcacgtt acaccacgat cacggcagca caagcgtgct atgaccgcat
2161 tatctactgt gataccgact caattcatct gaccggcacg gaaatcccgg atgtgattaa
2221 agatatcgtt gacccgaaaa aactgggtta ttgggcacac gaatcgacct ttaaacgtgc
2281 taaatacctg cgccagaaaa cgtacatcca agacatctac atgaaagaag tcgatggcaa
2341 actggtggaa ggttcaccgg atgactatac cgacattaaa ttttcggtga aatgcgccgg
2401 catgaccgat aaaatcaaaa aagaagtcac gttcgaaaat ttcaaagtgg gtttcagtcg
2461 caaaatgaaa ccgaaaccgg tgcaagttcc gggcggcgtt gtcctggtgg atgatacctt
2521 cacgattaaa cagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc
2581 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
2641 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
2701 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
2761 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
2821 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
2881 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
2941 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
3001 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
3061 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
3121 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
3181 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
3241 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
3301 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
3361 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
3421 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
3481 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
3541 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
3601 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
3661 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
3721 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
3781 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
3841 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
3901 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
3961 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
4021 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
4081 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
4141 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
4201 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
4261 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
4321 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
4381 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
4441 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
4501 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
4561 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
4621 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
4681 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
4741 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
4801 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
4861 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
4921 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
4981 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
5041 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
5101 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
5161 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
5221 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
5281 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
5341 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
5401 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
5461 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
5521 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
5581 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
5641 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
5701 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
5761 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
5821 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
5881 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
5941 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
6001 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
6061 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
6121 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
6181 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
6241 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
6301 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
6361 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
6421 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
6481 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
6541 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
6601 gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc
6661 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
6721 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
6781 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
6841 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
6901 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
6961 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
7021 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
7081 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
7141 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
7201 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
7261 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
7321 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
7381 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
7441 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
7501 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
7561 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
7621 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
7681 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
7741 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
7801 agatcgatct cgatcccgcg aaat
//
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