| 质粒名称: | pET21-10XHis-GST-HRV-dL5 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 73214 |
| 存储实验室: | Marcel Bruchez |
|
载体骨架基本信息 |
|
| 载体名称: | pET21a |
| 载体出品公司: | Novagen |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 5384 bp |
| 完整质粒大小: | 6500 bp |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | 低拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | EGFR,dL5** FAP |
| 物种来源: | H. sapiens (human); Schistosoma japonicum |
| 插入大小: | 1482 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | T7 |
| 标签/融合蛋白: | 10XHis GST HRV（插入件上的N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 and GSTSeqF |
| 3' 测序引物 | T7term |
|
文献引用方法 |
|
| 材料和方法部分: | pET21-10XHis-GST-HRV-dL5 was a gift from Marcel Bruchez (Addgene plasmid # 73214 ; http://n2t.net/addgene:73214 ; RRID:Addgene_73214) |
| 参考文献部分: | Fluorogen activating protein-affibody probes: modular, no-wash measurement of epidermal growth factor receptors. Wang Y, Telmer CA, Schmidt BF, Franke JD, Ort S, Arndt-Jovin DJ, Bruchez MP. Bioconjug Chem. 2015 Jan 21;26(1):137-44. doi: 10.1021/bc500525b. Epub 2014 Dec 19. 10.1021/bc500525b PubMed 25490520 |
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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 pET21-10XHis-GST-HRV-dL5 6868 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Bacterial expression vector for dL5(E52D) protein with N-terminal
His10, GST and HRV3C cleavage site (MBIC5, dL5**, FAP).
ACCESSION .
VERSION .
KEYWORDS pET21-10XHis-GST-HRV-dL5
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6868)
AUTHORS Wang Y, Telmer CA, Schmidt BF, Franke JD, Ort S, Arndt-Jovin DJ,
Bruchez MP
TITLE Fluorogen activating protein-affibody probes: modular, no-wash
measurement of epidermal growth factor receptors.
JOURNAL Bioconjug Chem. 2015 Jan 21;26(1):137-44. doi: 10.1021/bc500525b.
Epub 2014 Dec 19.
PUBMED 25490520
REFERENCE 2 (bases 1 to 6868)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6868
/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"
promoter 494..598
/gene="bla"
/label=AmpR promoter
CDS 599..1459
/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(817..836)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 1630..2218
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 2119..2138
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 2372..2389
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 2404..2546
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(2472..2491)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2632..2654
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(2648..2839)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(3648..4730)
/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 4692..4711
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(4731..4808)
/gene="lacI"
/label=lacI promoter
primer_bind 5014..5033
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 5117..5136
/label=T7
/note="T7 promoter, forward primer"
promoter 5117..5135
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 5136..5160
/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 5175..5197
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 5210..5239
/codon_start=1
/product="10xHis affinity tag"
/label=10xHis
/translation="HHHHHHHHHH"
CDS 5243..5896
/codon_start=1
/product="glutathione S-transferase from Schistosoma
japonicum"
/label=GST
/translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
primer_bind 5854..5876
/label=pGEX 5'
/note="pGEX vectors, Glutathione-S-transferase, forward
primer"
CDS 5918..5941
/codon_start=1
/product="recognition and cleavage site for human
rhinovirus 3C and PreScission proteases"
/label=HRV 3C site
/translation="LEVLFQGP"
CDS 6712..6729
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(6782..6800)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 6796..6843
/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 attaacgttt acaatttcag gtggcacttt
481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
541 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
601 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
661 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
721 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
781 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
841 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
901 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
961 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
1021 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
1081 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
1141 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
1201 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
1261 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
1321 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
1381 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
1441 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
1501 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
1561 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
1621 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
1681 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
1741 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
1801 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
1861 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
1921 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
1981 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
2041 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
2101 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
2161 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
2221 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
2281 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
2341 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
2401 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg
2461 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
2521 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
2581 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
2641 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
2701 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
2761 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
2821 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
2881 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
2941 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
3001 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
3061 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
3121 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
3181 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
3241 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
3301 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
3361 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
3421 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
3481 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
3541 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
3601 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
3661 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
3721 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
3781 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
3841 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
3901 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
3961 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
4021 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
4081 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
4141 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
4201 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
4261 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
4321 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
4381 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
4441 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
4501 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
4561 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
4621 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
4681 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
4741 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
4801 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
4861 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
4921 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
4981 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
5041 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
5101 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
5161 cctctagaaa taattttgtt taactttaag aaggagatat acatatatgc accatcatca
5221 ccaccatcat caccaccata gcatgtcccc tatactaggt tattggaaaa ttaagggcct
5281 tgtgcaaccc actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta
5341 tgagcgcgat gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc
5401 caatcttcct tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg
5461 ttatatagct gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc
5521 aatgcttgaa ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa
5581 agactttgaa actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt
5641 cgaagatcgt ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt
5701 catgttgtat gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt
5761 cccaaaatta gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt
5821 gaaatccagc aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg
5881 tgaccatcct ccaaaatcgg attctccgaa ttgcggcctg gaagttctgt tccagggtcc
5941 gggatccctt gaattccagg ccgtcgttac ccaagaacct agtgttaccg ttagcccagg
6001 tggtactgtt atacttactt gtggaagtgg tacgggtgcc gtcacatctg gtcattatgc
6061 aaattggttt caacaaaaac caggacaagc tccaagagct ttgatttttg atactgataa
6121 gaagtattct tggaccccag gtagattttc tggatctttg ctgggagcaa aggcagcttt
6181 gacaatatca gatgctcagc ctgaggacga agccgagtat tactgttctc ttagcgacgt
6241 ggatggctac ttgtttggcg gtggaacaca actgacggtt ctgtccggtg gtggcggctc
6301 tggtggcggt ggcagcggcg gtggtggttc cggaggcggc ggttctcagg ctgtggtgac
6361 tcaggagccg tcagtgactg tgtccccagg agggacagtc attctcactt gtggctccgg
6421 cactggagct gtcaccagtg gtcattatgc caactggttc cagcagaagc ctggccaagc
6481 ccccagggca cttatatttg acaccgacaa gaagtattcc tggacccctg gccgattctc
6541 aggctccctc cttggggcca aggctgccct gaccatctcg gatgcgcagc ctgaagatga
6601 ggctgagtat tactgttcgc tctccgacgt tgacggttat ctgttcggag gaggcaccca
6661 gctgaccgtc ctctccaccg gtatcttact cgagtgaatg aatgagtcga gcaccaccac
6721 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
6781 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
6841 ttgctgaaag gaggaactat atccggat
//
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