| 质粒名称: | pET_RTX(exo-)-6xHis |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 145028 |
| 存储实验室: | Andrew Ellington |
|
载体骨架基本信息 |
|
| 载体名称: | modified pET21 |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 5871 bp |
| 完整质粒大小: | 8232 bp |
| 载体类型: | 细菌表达 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | RTX(exo-),Exonuclease deficient variant of RTX |
| 物种来源: | Synthetic |
| 插入大小: | 2361 bp |
| 基因突变: | N210D |
| 启动子 | T7 |
| 标签/融合蛋白: | 6xHis（插入件上的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(exo-)-6xHis was a gift from Andrew Ellington (Addgene plasmid # 145028 ; http://n2t.net/addgene:145028 ; RRID:Addgene_145028) |
| 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, Ellington AD. bioRxiv 10.1101/2020.03.29.013342 |
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
-6xHis质粒图谱.png)
载体简介
载体序列
LOCUS pET_RTX(exo-)-6xHis 8232 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION pET vector for IPTG-inducible, T7-based expression of the
exonuclease deficient version of RTX bearing a C-terminal 6xHis tag.
ACCESSION .
VERSION .
KEYWORDS pET_RTX(exo-)-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 40..126
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 223..309
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 406..492
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 589..675
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 772..858
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
primer_bind 993..1012
/label=T7
/note="T7 promoter, forward primer"
promoter 993..1011
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 1012..1036
/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 1051..1073
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
RBS 3009..3017
/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 3418..3435
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(3502..3520)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 3516..3563
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter 3623..3727
/gene="bla"
/label=AmpR promoter
CDS 3728..4588
/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(3946..3965)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin complement(4630..5143)
/direction=LEFT
/label=M13 ori
/note="M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4825..4846)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 5037..5056
/label=F1ori-R
/note="F1 origin, reverse primer"
rep_origin 5254..5842
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5743..5762
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5996..6013
/label=L4440
/note="L4440 vector, forward primer"
primer_bind complement(6035..6054)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 6195..6217
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(6211..6402)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(6959..8041)
/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 8003..8022
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(8042..8119)
/gene="lacI"
/label=lacI promoter
ORIGIN
1 ctttcgtctt caagaattaa ttcccaattc cccaggcatc aaataaaacg aaaggctcag
61 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
121 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
181 ggacgcccgc cataaactgc caggaattaa ttccccaggc atcaaataaa acgaaaggct
241 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
301 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
361 gcaggacgcc cgccataaac tgccaggaat taattcccca ggcatcaaat aaaacgaaag
421 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
481 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
541 cgggcaggac gcccgccata aactgccagg aattaattcc ccaggcatca aataaaacga
601 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
661 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
721 tggcgggcag gacgcccgcc ataaactgcc aggaattaat tccccaggca tcaaataaaa
781 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
841 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
901 gggtggcggg caggacgccc gccataaact gccaggaatt ggggatcgga attaattccc
961 ggtttaaacc ggggatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
1021 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
1081 atgattcttg acacggatta catcacggaa gacggcaagc cggttatccg tattttcaag
1141 aaagaaaacg gcgaattcaa gattgaatac gatcggacat ttgaaccgta cctgtacgct
1201 ctcctcaagg atgatagcgc aatcgaagaa gtgaaaaaaa tcaccgcaga gcggcatggc
1261 acagtggtaa cagttaagcg ggtcgagaaa gtgcagaaga agttcttagg ccggccagtc
1321 gaagtatgga aattatactt cacacatcca caggacgttc cggcgatcat ggataagatt
1381 cgggagcatc cggcggtaat cgatatctat gaatacgata ttccgttcgc tattcgctac
1441 cttattgaca aaggtttagt tccaatggag ggtgatgagg aacttaaact gttagcattc
1501 gatatcgaaa cactttatca cgaaggtgaa gagtttgccg aaggtccgat tttaatgatc
1561 tcgtacgccg atgaagaagg cgcacgcgta attacgtgga aaaatgtgga cctcccgtac
1621 gtagacgtag tgagcactga gcgcgagatg attaaacgtt tccttcgggt agtaaaagaa
1681 aaagacccag acgtgctgat tacgtatgac ggcgacaact ttgattttgc ctatctcaag
1741 aagcgttgcg aaaagttagg cattaatttc gccctgggtc gggacggttc agagccgaaa
1801 attcagcgga tgggcgaccg ctttgctgtg gaggtaaaag gtcgcatcca tttcgattta
1861 tatccggtta tccggcgcac catcaacttg ccgacttaca cacttgaagc agtttacgaa
1921 gcggtgttcg gccaaccaaa agaaaaggtt tatgccgagg agattaccac cgcatgggaa
1981 actggcgaaa acttggagcg ggtggctcgg tattccatgg aagatgccaa ggtgacctac
2041 gaactgggca aagagttttt accgatggaa gcacaattaa gccgccttat tggtcagtcc
2101 ctctgggatg tgtcgcgttc ttcaacgggc aatttagtcg aatggtttct tcttcggaaa
2161 gcgtacgagc gtaacgagct tgctccaaat aagccagacg aaaaagaatt ggctcggcgc
2221 catcagtcac atgagggcgg ctacattaag gagccagaac ggggcttgtg ggagaacatc
2281 gtctaccttg attttcggtc tctttatccg tctattatca tcacacataa cgtctcgcca
2341 gataccctga accgtgaagg ctgtaaagaa tatgatgtgg caccacaggt cggccatcgt
2401 ttttgtaaag acttcccggg cttcattcca tctcttctgg gtgatttgtt agaagagcgt
2461 caaaagatca agaaacgtat gaaagcgaca attgacccaa ttgaacgcaa attacttgat
2521 taccgtcagc gtgcaatcaa gatcctcgcg aactctctgt acggttatta cggctacgca
2581 cgcgcccggt ggtattgcaa agaatgtgca gaatcagtca ttgcttgggg tcgggagtac
2641 ctgaccatga cgattaagga aattgaggag aaatacggtt tcaaggtcat ctatagtgac
2701 acggatggtt tctttgcaac gattccaggt gcggacgcag aaactgtaaa gaaaaaggca
2761 atggagttct tgaagtatat taatgcgaag ttgccaggcg ccctggaatt agagtacgaa
2821 ggtttttata agcgtggcct gttcgtgaca aagaagaaat acgcggtaat tgacgaggaa
2881 ggcaagatca caactcgtgg cttggaaatt gttcgtcgcg attggagcga gatcgcaaag
2941 gagacccaag ctcgtgtgtt ggaggccctc ctgaaggatg gtgacgtcga aaaagccgta
3001 cgcatcgtta aggaggttac agagaagctt agcaagtatg aggtcccacc agagaaactt
3061 gttattcata aacaaatcac tcgcgacctt aaagactata aggccactgg tccacacgtc
3121 gccgtagcaa agcggcttgc ggctcggggc gtcaagattc ggccaggcac ggttattagt
3181 tacatcgtcc tcaaaggctc aggccggatt gttgatcgcg cgattccatt tgatgaattt
3241 gatccgacga agcataaata tgatgcggaa tattacattg aaaaacaggt tctgccggcg
3301 gtggagcgca tcttacgtgc gttcggctat cgcaaggagg atttgcggta ccagaaaact
3361 cgtcaagtcg gtttgagtgc ccgtctgaag ccgaaaggta ccctcgaggg atccagccat
3421 caccatcacc atcactgata agcggccgca ttgatccggc tgctaacaaa gcccgaaagg
3481 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
3541 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggataa ctacgtcagg
3601 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
3661 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
3721 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
3781 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
3841 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
3901 tcgccccgaa gaacgtttcc caatgatgag cacttttaaa gttctgctat gtggcgcggt
3961 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
4021 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
4081 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
4141 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
4201 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
4261 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
4321 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
4381 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
4441 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
4501 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
4561 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
4621 gattgattta ccccggttga taatcagaaa agccccaaaa acaggaagat tgtataagca
4681 aatatttaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
4741 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
4801 cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
4861 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
4921 tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
4981 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
5041 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
5101 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtaaaagga tctaggtgaa
5161 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
5221 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
5281 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
5341 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
5401 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
5461 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
5521 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
5581 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
5641 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
5701 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
5761 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
5821 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
5881 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
5941 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
6001 gtcagtgagc gaggaagcta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
6061 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
6121 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
6181 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
6241 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac agatgtctgc
6301 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
6361 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
6421 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
6481 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
6541 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagccga acgccagcaa
6601 gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
6661 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
6721 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
6781 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
6841 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
6901 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
6961 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
7021 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
7081 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
7141 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
7201 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
7261 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
7321 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
7381 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
7441 agccagccag acgcggacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
7501 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
7561 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
7621 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
7681 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
7741 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
7801 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
7861 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
7921 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
7981 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
8041 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
8101 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
8161 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
8221 cggcatgccg cc
//
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