| 质粒名称: | pETNKI-strepII-3C-LIC-kan |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 108706 |
| 存储实验室: | Titia Sixma |
|
载体骨架基本信息 |
|
| 载体名称: | pET |
| 载体出品公司: | Novagen |
| 空载体大小: | 5513 bp |
| 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: | 卡那霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 低拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | -- |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | T7 |
| 标签/融合蛋白: | StrepII-3C蛋白酶切割位点（插入物上的N端） |
| 克隆方法: | Ligation Independent克隆 |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | T7 |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pETNKI-strepII-3C-LIC-kan was a gift from Titia Sixma (Addgene plasmid # 108706 ; http://n2t.net/addgene:108706 ; RRID:Addgene_108706) |
| 参考文献部分: | Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases. Luna-Vargas MP, Christodoulou E, Alfieri A, van Dijk WJ, Stadnik M, Hibbert RG, Sahtoe DD, Clerici M, Marco VD, Littler D, Celie PH, Sixma TK, Perrakis A. J Struct Biol. 2011 Aug;175(2):113-9. doi: 10.1016/j.jsb.2011.03.017. Epub 2011 Mar 29. 10.1016/j.jsb.2011.03.017 PubMed 21453775 |
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 pETNKI-strepII-3C-LIC-kan 5309 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Expression of a StrepII-tagged target protein with 3C protease
cleavage site..
ACCESSION .
VERSION .
KEYWORDS pETNKI-strepII-3C-LIC-kan
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5309)
AUTHORS Luna-Vargas MP, Christodoulou E, Alfieri A, van Dijk WJ, Stadnik M,
Hibbert RG, Sahtoe DD, Clerici M, Marco VD, Littler D, Celie PH,
Sixma TK, Perrakis A
TITLE Enabling high-throughput ligation-independent cloning and protein
expression for the family of ubiquitin specific proteases.
JOURNAL J Struct Biol. 2011 Aug;175(2):113-9. doi:
10.1016/j.jsb.2011.03.017. Epub 2011 Mar 29.
PUBMED 21453775
REFERENCE 2 (bases 1 to 5309)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..5309
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 7..462
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(94..113)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 304..325
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(554..1369)
/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 1277..1296
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 1361..1380
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 1491..2079
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1980..1999
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 2233..2250
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 2265..2404
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(2330..2349)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2490..2512
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(2506..2697)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(3506..4588)
/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 4550..4569
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(4589..4666)
/gene="lacI"
/label=lacI promoter
primer_bind 4872..4891
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 4975..4994
/label=T7
/note="T7 promoter, forward primer"
promoter 4975..4993
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 4994..5018
/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 5033..5055
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 5071..5094
/codon_start=1
/product="peptide that binds Strep-Tactin(R), an engineered
form of streptavidin"
/label=Strep-Tag II
/translation="WSHPQFEK"
CDS 5101..5124
/codon_start=1
/product="recognition and cleavage site for human
rhinovirus 3C and PreScission proteases"
/label=HRV 3C site
/translation="LEVLFQGP"
CDS 5148..5165
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(5218..5236)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 5232..5279
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
ORIGIN
1 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
61 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
121 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
181 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
241 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
301 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
361 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
421 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
481 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
541 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
601 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
661 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
721 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
781 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
841 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
901 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
961 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
1021 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
1081 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
1141 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
1201 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
1261 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
1321 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
1381 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
1441 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
1501 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
1561 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
1621 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
1681 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
1741 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
1801 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
1861 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
1921 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
1981 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
2041 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
2101 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
2161 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
2221 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
2281 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
2341 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
2401 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
2461 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
2521 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
2581 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
2641 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
2701 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
2761 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
2821 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
2881 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
2941 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
3001 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
3061 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
3121 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
3181 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
3241 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
3301 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
3361 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
3421 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
3481 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
3541 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
3601 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
3661 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
3721 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
3781 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
3841 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
3901 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
3961 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
4021 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
4081 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
4141 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
4201 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
4261 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
4321 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
4381 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
4441 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
4501 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
4561 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
4621 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
4681 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
4741 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
4801 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
4861 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
4921 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
4981 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
5041 actttaagaa ggagatatac catggcaagc tggagccacc cgcagttcga aaagggtgca
5101 cttgaagtcc tctttcaggg acccgggtac ccgggcttct cctcgagcac caccaccacc
5161 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
5221 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
5281 tgaaaggagg aactatatcc ggattggcg
//
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