| 质粒名称: | pET_RTX-6xHis |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 145029 |
| 存储实验室: | Andrew Ellington |
|
载体骨架基本信息 |
|
| 载体名称: | modified pET21 |
| 空载体大小: | 5871 bp |
| 完整质粒大小: | 8232 bp |
| 载体类型: | 细菌表达 |
| 载体抗性: | 氨苄霉素100 μg/mL |
| 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: | RTX,Reverse Transcription Xenopolymerase |
| 物种来源: | Synthetic |
| 插入大小: | 2361 bp |
| 启动子: | T7 |
| 标签/融合蛋白: | 6xHis（插件上的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: | NotI (未破坏) |
| 5’测序引物 | GATCTCGATCCCGCGAAATTAATACGAC |
| 3’测序引物 | TTGCTCAGCGGTGGCAGCAGC |
|
文献引用方法 |
|
| 材料和方法部分: | pET_RTX-6xHis was a gift from Andrew Ellington (Addgene plasmid # 145029 ; http://n2t.net/addgene:145029 ; RRID:Addgene_145029) |
| 参考文献部分: | A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production. Bhadra S, Maranhao AC, Paik I, Ellington AD. BioProtocol 2021 10.21769/BioProtoc.3898 |
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_RTX-6xHis 8232 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION pET vector for IPTG-inducible, T7-based expression of RTX bearing a
C-terminal 6xHis tag.
ACCESSION .
VERSION .
KEYWORDS pET_RTX-6xHis
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8232)
AUTHORS Bhadra S, Maranhao AC, Ellington AD
TITLE A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for
reagent production
JOURNAL bioRxiv
REFERENCE 2 (bases 1 to 8232)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8232
/organism="synthetic DNA construct"
/mol_type="other DNA"
terminator 86..172
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 269..355
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 452..538
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 635..721
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 818..904
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
primer_bind 1039..1058
/label=T7
/note="T7 promoter, forward primer"
promoter 1039..1057
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 1058..1082
/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 1097..1119
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
RBS 3055..3063
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 3464..3481
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(3548..3566)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 3562..3609
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter 3669..3773
/gene="bla"
/label=AmpR promoter
CDS 3774..4634
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(3992..4011)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin complement(4676..5189)
/direction=LEFT
/label=M13 ori
/note="M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4871..4892)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 5083..5102
/label=F1ori-R
/note="F1 origin, reverse primer"
rep_origin 5300..5888
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5789..5808
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 6042..6059
/label=L4440
/note="L4440 vector, forward primer"
primer_bind complement(6081..6100)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 6241..6263
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(6257..6448)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(7005..8087)
/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
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARPRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
primer_bind 8049..8068
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(8088..8165)
/gene="lacI"
/label=lacI promoter
ORIGIN
1 gttgagcacc gccgccgcaa ggaatggtgc atgccggcat gccgcccttt cgtcttcaag
61 aattaattcc caattcccca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
121 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
181 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata
241 aactgccagg aattaattcc ccaggcatca aataaaacga aaggctcagt cgaaagactg
301 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
361 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
421 ataaactgcc aggaattaat tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga
481 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
541 gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc
601 gccataaact gccaggaatt aattccccag gcatcaaata aaacgaaagg ctcagtcgaa
661 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
721 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
781 cccgccataa actgccagga attaattccc caggcatcaa ataaaacgaa aggctcagtc
841 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
901 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg
961 acgcccgcca taaactgcca ggaattgggg atcggaatta attcccggtt taaaccgggg
1021 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
1081 cccctctaga aataattttg tttaacttta agaaggagat atacatatga ttcttgacac
1141 ggattacatc acggaagacg gcaagccggt tatccgtatt ttcaagaaag aaaacggcga
1201 attcaagatt gaatacgatc ggacatttga accgtacctg tacgctctcc tcaaggatga
1261 tagcgcaatc gaagaagtga aaaaaatcac cgcagagcgg catggcacag tggtaacagt
1321 taagcgggtc gagaaagtgc agaagaagtt cttaggccgg ccagtcgaag tatggaaatt
1381 atacttcaca catccacagg acgttccggc gatcatggat aagattcggg agcatccggc
1441 ggtaatcgat atctatgaat acgatattcc gttcgctatt cgctacctta ttgacaaagg
1501 tttagttcca atggagggtg atgaggaact taaactgtta gcattcgata tcgaaacact
1561 ttatcacgaa ggtgaagagt ttgccgaagg tccgatttta atgatctcgt acgccgatga
1621 agaaggcgca cgcgtaatta cgtggaaaaa tgtggacctc ccgtacgtag acgtagtgag
1681 cactgagcgc gagatgatta aacgtttcct tcgggtagta aaagaaaaag acccagacgt
1741 gctgattacg tataacggcg acaactttga ttttgcctat ctcaagaagc gttgcgaaaa
1801 gttaggcatt aatttcgccc tgggtcggga cggttcagag ccgaaaattc agcggatggg
1861 cgaccgcttt gctgtggagg taaaaggtcg catccatttc gatttatatc cggttatccg
1921 gcgcaccatc aacttgccga cttacacact tgaagcagtt tacgaagcgg tgttcggcca
1981 accaaaagaa aaggtttatg ccgaggagat taccaccgca tgggaaactg gcgaaaactt
2041 ggagcgggtg gctcggtatt ccatggaaga tgccaaggtg acctacgaac tgggcaaaga
2101 gtttttaccg atggaagcac aattaagccg ccttattggt cagtccctct gggatgtgtc
2161 gcgttcttca acgggcaatt tagtcgaatg gtttcttctt cggaaagcgt acgagcgtaa
2221 cgagcttgct ccaaataagc cagacgaaaa agaattggct cggcgccatc agtcacatga
2281 gggcggctac attaaggagc cagaacgggg cttgtgggag aacatcgtct accttgattt
2341 tcggtctctt tatccgtcta ttatcatcac acataacgtc tcgccagata ccctgaaccg
2401 tgaaggctgt aaagaatatg atgtggcacc acaggtcggc catcgttttt gtaaagactt
2461 cccgggcttc attccatctc ttctgggtga tttgttagaa gagcgtcaaa agatcaagaa
2521 acgtatgaaa gcgacaattg acccaattga acgcaaatta cttgattacc gtcagcgtgc
2581 aatcaagatc ctcgcgaact ctctgtacgg ttattacggc tacgcacgcg cccggtggta
2641 ttgcaaagaa tgtgcagaat cagtcattgc ttggggtcgg gagtacctga ccatgacgat
2701 taaggaaatt gaggagaaat acggtttcaa ggtcatctat agtgacacgg atggtttctt
2761 tgcaacgatt ccaggtgcgg acgcagaaac tgtaaagaaa aaggcaatgg agttcttgaa
2821 gtatattaat gcgaagttgc caggcgccct ggaattagag tacgaaggtt tttataagcg
2881 tggcctgttc gtgacaaaga agaaatacgc ggtaattgac gaggaaggca agatcacaac
2941 tcgtggcttg gaaattgttc gtcgcgattg gagcgagatc gcaaaggaga cccaagctcg
3001 tgtgttggag gccctcctga aggatggtga cgtcgaaaaa gccgtacgca tcgttaagga
3061 ggttacagag aagcttagca agtatgaggt cccaccagag aaacttgtta ttcataaaca
3121 aatcactcgc gaccttaaag actataaggc cactggtcca cacgtcgccg tagcaaagcg
3181 gcttgcggct cggggcgtca agattcggcc aggcacggtt attagttaca tcgtcctcaa
3241 aggctcaggc cggattgttg atcgcgcgat tccatttgat gaatttgatc cgacgaagca
3301 taaatatgat gcggaatatt acattgaaaa acaggttctg ccggcggtgg agcgcatctt
3361 acgtgcgttc ggctatcgca aggaggattt gcggtaccag aaaactcgtc aagtcggttt
3421 gagtgcccgt ctgaagccga aaggtaccct cgagggatcc agccatcacc atcaccatca
3481 ctgataagcg gccgcattga tccggctgct aacaaagccc gaaaggaagc tgagttggct
3541 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
3601 ggttttttgc tgaaaggagg aactatatcc ggataactac gtcaggtggc acttttcggg
3661 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
3721 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
3781 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
3841 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
3901 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
3961 gtttcccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
4021 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
4081 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
4141 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
4201 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
4261 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
4321 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
4381 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
4441 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
4501 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
4561 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
4621 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttacccc
4681 ggttgataat cagaaaagcc ccaaaaacag gaagattgta taagcaaata tttaaattgt
4741 aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
4801 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagcccg agatagggtt
4861 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
4921 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac ccaaatcaag
4981 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
5041 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
5101 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
5161 cgcgcttaat gcgccgctac agggcgcgta aaaggatcta ggtgaagatc ctttttgata
5221 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
5281 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
5341 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
5401 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
5461 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
5521 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
5581 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
5641 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
5701 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
5761 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
5821 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
5881 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
5941 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
6001 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
6061 aagctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
6121 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
6181 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
6241 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
6301 ggtaaagctc atcagcgtgg tcgtgcagcg attcacagat gtctgcctgt tcatccgcgt
6361 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
6421 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
6481 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
6541 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
6601 accagagaaa aatcactcag ggtcaatgcc agccgaacgc cagcaagacg tagcccagcg
6661 cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
6721 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
6781 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
6841 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
6901 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
6961 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
7021 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
7081 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
7141 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
7201 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
7261 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
7321 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
7381 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
7441 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
7501 ggacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
7561 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
7621 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
7681 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
7741 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
7801 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
7861 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
7921 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
7981 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
8041 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
8101 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
8161 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
8221 taggttgagg cc
//
