| 质粒名称: | pET-NT*-HRV3CP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 162795 |
| 存储实验室: | Gottfried Otting |
|
载体骨架基本信息 |
|
| 载体名称: | pET-47b(+) |
| 载体出品公司: | Clontech |
| 空载体大小: | 5011 bp |
| 完整质粒大小: | 6004 bp |
| 载体类型: | 大肠杆菌表达 |
| 载体抗性: | 卡那霉素 |
| 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: | Human rhinovirus 14 3C protease |
| 基因又名: | NT*-HRV3CP |
| 物种来源: | Human rhinovirus 14 3C |
| 插入大小: | 993 bp |
| 基因突变: | T2A, S13L, A92T, V104T, V114I, E161K, Y193K, F198Y, D202T, I203V, Y258F, A283V, K288T, N290Y relative to RpBphP2 |
| GenBank编号 | NP_740524.1 |
| 启动子: | T7 |
| 标签/融合蛋白: | 人细胞色素c氧化酶亚基VIII（N端） |
| 克隆方法: | Ligation Independent Cloning |
| 5’克隆位点: | AgeI(未破坏) |
| 3’克隆位点: | NotI (未破坏) |
| 5’测序引物： | TAATACGACTCACTATAGGG |
| 3’测序引物 | GCTAGTTATTGCTCAGCGG |
|
文献引用方法 |
|
| 材料和方法部分: | pET-NT*-HRV3CP was a gift from Gottfried Otting (Addgene plasmid # 162795 ; http://n2t.net/addgene:162795 ; RRID:Addgene_162795) |
| 参考文献部分: | NT*-HRV3CP: an optimized construct of human rhinovirus 14 3C protease for high-yield expression and fast affinity-tag cleavage. Abdelkader EH, Otting G. J Biotechnol. 2020 Nov 6. pii: S0168-1656(20)30306-0. doi: 10.1016/j.jbiotec.2020.11.005. 10.1016/j.jbiotec.2020.11.005 PubMed 33166527 |
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 pET-NT*-HRV3CP 6000 bp ds-DNA circular SYN 06-JUL-2023
DEFINITION Expressess an optimized construct of human rhinovirus 14 3C protease
(NT*-HRV3CP).
ACCESSION .
VERSION .
KEYWORDS pET-NT*-HRV3CP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6000)
AUTHORS Abdelkader EH, Otting G
TITLE NT*-HRV3CP: an optimized construct of human rhinovirus 14 3C
protease for high-yield expression and fast affinity-tag cleavage.
JOURNAL J Biotechnol. 2020 Nov 6. pii: S0168-1656(20)30306-0. doi:
10.1016/j.jbiotec.2020.11.005.
PUBMED 33166527
REFERENCE 2 (bases 1 to 6000)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jul 6, 2023 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6000
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 1029..1220
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
primer_bind complement(1214..1236)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
misc_feature 1322..1461
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 1377..1396
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind complement(1476..1493)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(1647..2235)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(1727..1746)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind complement(2346..2365)
/label=pENTR-R
/note="pENTR vectors, reverse primer"
CDS 2357..3172
/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 complement(2430..2449)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
rep_origin complement(3264..3719)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3401..3422)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 3613..3632
/label=F1ori-R
/note="F1 origin, reverse primer"
terminator 3756..3803
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
primer_bind 3799..3817
/label=T7 Term
/note="T7 terminator, reverse primer"
CDS complement(4876..4893)
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
RBS 4904..4926
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind 4941..4965
/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)."
primer_bind complement(4965..4984)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(4966..4984)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(5072..5091)
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
promoter 5297..5374
/gene="lacI"
/label=lacI promoter
CDS 5375..457
/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 complement(5394..5413)
/label=LacI-R
/note="LacI, reverse primer"
ORIGIN
1 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
61 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
121 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggacatctc
181 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
241 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
301 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
361 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
421 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
481 agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca
541 gctccttccg gtgggcgcgg ggcatgacta gcatgatcgt gctcctgtcg ttgaggaccc
601 ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa
661 cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg
721 gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc
781 ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga
841 agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt
901 gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga
961 gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg
1021 aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc
1081 agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct
1141 gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg
1201 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
1261 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
1321 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
1381 atcagagcag attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta
1441 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
1501 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
1561 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
1621 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
1681 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
1741 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
1801 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
1861 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
1921 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
1981 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
2041 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
2101 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
2161 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
2221 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
2281 gaaaactcac gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag
2341 taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt
2401 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
2461 atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa
2521 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
2581 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
2641 gttactcacc actgcgatcc ccggcaaaac agcattccag gtattagaag aatatcctga
2701 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
2761 tgtttgtaat tgtcctttta acagtgatcg cgtatttcgt ctcgctcagg cgcaatcacg
2821 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
2881 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
2941 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
3001 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
3061 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
3121 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat
3181 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
3241 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
3301 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
3361 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
3421 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
3481 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
3541 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
3601 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
3661 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
3721 ccattcgcca atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta
3781 gaggccccaa ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc
3841 tttcgggctt tgttagcagc cggatctatc gatgcatgcc atggtacccg ggagctcgaa
3901 ttcttactgc ttttccacga aatactgttt tttcagctgt gcgctaaaac cctgacgacc
3961 attgccacca acatgaatgc caaagatttt accggttgca cacagaacac caccacactg
4021 accggttttg gttgcataat cataacgaat catacgattg gtcggggtac tgctcagatt
4081 aatcagacct gccatggtaa ccggaccaac ttccaggatg gtgttggtaa agttattgct
4141 atgaacaacc agggttgcat caacaccttc cagatcttcg ctgataaagc cacggatatc
4201 acgaaatttt tcattacgat ccagggtcag aacggtcagt tccagattaa tgttttccgg
4261 atccaccagt ttatacttgt ctttcacgcg aattttctga ccattaacca gaacgtcatc
4321 acccggttgt gcatgtgtcg gaataacaca aacacgatca tgaataccca gaccggtaaa
4381 ttcgcctttg gaggtggtaa tggtcataat gtttttacgc agcaggctca gtgcaaattc
4441 ggtattcgga ccgctaccac caccaccgct attacctgcg ctaacatcat tcatacctgc
4501 ctgtgcaaac atgctaacca gctgggtaat ttcgttaata aacggctgat taacaacacc
4561 ggttgtctgc agaaatgcat tgctcattgc gcttgcaatg ctgctggttt tggtgctcag
4621 gctaccgcca ccttcttcgc ttgctgcaat ttctgccatg ctgcttgcaa atgccatatt
4681 cagtgcctgc agatcattcg gactggtacg accctgtgct gccaggctct gaatgctctg
4741 aaccatgctc tgtgcaatgg tgctcatttt gtccagctgg cttgcggtaa aaccaggcat
4801 gctgctcaga ccctgcataa agctattcat aaagttttct gccagaccag gattggtcca
4861 cggtgtggta tggctgtggt gatgatgatg atgcatatgt atatctcctt cttaaagtta
4921 aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt
4981 attaatttcg cgggatcgag atcgatctcg atcctctacg ccggacgcat cgtggccggc
5041 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
5101 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
5161 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg
5221 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
5281 gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
5341 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
5401 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
5461 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
5521 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
5581 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
5641 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
5701 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
5761 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
5821 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
5881 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
5941 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
//
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