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pETDuet-1 构建费用500元/个起

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基本信息

别名

pETDuet1, pETDuet 1, pETDuet-1

体类型

大肠杆菌蛋白双表达载体,有两个多克隆位点(双启动子)

载体抗性

氨苄青霉素

载体标签

N-His, C-S

筛选标记

/

启动子

T7lac

表达水平

高拷贝

载体大小

5420bp

克隆方法

多克隆位点,限制性内切酶

5' 测序引物

DUETF1: TGGTGTCCGGGATCTCGA
DUETF2: TTGTACACGGCCGCATAATC

3' 测序引物

DUETR1: 5'-GATTATGCGGCCGTGTACAA-3' 
T7 ter: TGCTAGTTATTGCTCAGCGG

组成型

组成型

备注

pETDuet-1是设计用来同时表达两个目的基因。pETDuet-1携带有pBR322质粒来源的ColE1复制子, lacI基因和氨苄抗性基因。

Bacterial vector for the co-expression of two genes.

质粒图谱和多克隆位点信息

注意:

客户在我公司选择构建载体服务项目时,请自行提供母源载体。

如要使用本公司保存的母源载体时,本公司不能保证所有母源载体完全符合客户预期,也不承担由此产生的任何责任。

 

 

 

载体描述

pETDuet-1载体被设计用来在大肠杆菌中同时表达两个外源基因. 载体含有两个多克隆位点,每一个多克隆位点是通过T7启动子/Lac操纵子和一个核糖体结合位点组成。pETDuet-1携带有pBR322质粒来源的ColE1复制子, lacI基因和氨苄抗性基因。

本载体可以与pACYCDuet-1载体联合使用,在合适的宿主菌中,可以一起共表达至多4个目的基因。插入在pETDuet-1载体多克隆位点1的目的基因可以使用pET Upstream Primer (Cat. No. 69214-3)DuetDOWN1 Primer (Cat. No. 71179-3)进行测序,插入在多克隆位点2的目的基因可以通过DuetUP2 Primer (Cat. No. 71180-3)T7 Terminator Primer (Cat. No. 69337-3)进行目的基因测序。

 

pETDuet™-1 is designed for the coexpression of two target genes. The vector contains two multiple cloning sites (MCS), each of which is preceded by a T7 promoter/lac operator and a ribosome binding site (rbs). The vector also carries the pBR322-derived

ColE1 replicon, lacI gene and ampicillin resistance gene. This vector can be used in combination with pACYCDuet™-1 (Cat. No. 71147-3) in an appropriate host strain for the coexpression of up to 4 target genes. Genes inserted into MCS1 can be sequenced using the pET Upstream Primer (Cat. No. 69214-3) and DuetDOWN1 Primer (Cat. No. 71179-3). Genes inserted into MCS2 can be sequenced using the DuetUP2 Primer (Cat. No. 71180-3) and T7 Terminator Primer (Cat. No. 69337-3)

 

载体序列

LOCUS       pETDuet-1                5420 bp ds-DNA     circular SYN 23-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    pETDuet-1

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 5420)

  AUTHORS   BT-Lab

  TITLE     Direct Submission

  JOURNAL   Exported Saturday, Mar 23, 2019 from SnapGene Viewer 4.3.4

FEATURES             Location/Qualifiers

     source          1..5420

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     RBS             38..60

                     /note="efficient ribosome binding site from bacteriophage

                     T7 gene 10 (Olins and Rangwala, 1989)"

     CDS             79..96

                     /codon_start=1

                     /product="6xHis affinity tag"

                     /label=6xHis

                     /translation="HHHHHH"

     misc_feature    102..191

                     /label=MCS1

                     /note="multiple cloning site"

     primer_bind     complement(185..204)

                     /label=Down1 primer

     primer_bind     185..204

                     /label=UP2 primer

     promoter        210..228

                     /label=T7 promoter

                     /note="promoter for bacteriophage T7 RNA polymerase"

     protein_bind    229..253

                     /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)."

     misc_feature    293..355

                     /label=MCS2

                     /note="multiple cloning site"

     CDS             362..406

                     /codon_start=1

                     /product="affinity and epitope tag derived from pancreatic

                     ribonuclease A"

                     /label=S-Tag

                     /translation="KETAAAKFERQHMDS"

     primer_bind     complement(444..462)

                     /label=T7 ter primer

     terminator      458..505

                     /label=T7 terminator

                     /note="transcription terminator for bacteriophage T7 RNA

                     polymerase"

     rep_origin      542..997

                     /direction=RIGHT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow

                     indicates direction of (+) strand synthesis"

     CDS             complement(1112..1972)

                     /codon_start=1

                     /gene="bla"

                     /product="beta-lactamase"

                     /label=AmpR

                     /note="confers resistance to ampicillin, carbenicillin, and

                     related antibiotics"

                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI

                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS

                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW

                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA

                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS

                     LIKHW"

     promoter        complement(1973..2065)

                     /gene="bla"

                     /label=AmpR promoter

     rep_origin      2146..2734

                     /direction=RIGHT

                     /label=ori

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of

                     replication"

     misc_feature    2920..3062

                     /label=bom

                     /note="basis of mobility region from pBR322"

     CDS             complement(3164..3355)

                     /codon_start=1

                     /gene="rop"

                     /product="Rop protein, which maintains plasmids at low copy

                     number"

                     /label=rop

                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA

                     DELYRSCLARFGDDGENL"

     CDS             complement(3927..5009)

                     /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"

     promoter        complement(5010..5087)

                     /gene="lacI"

                     /label=lacI promoter

     primer_bind     5354..5369

                     /label=UP1 primer

     promoter        5400..5418

                     /label=T7 promoter

     protein_bind    join(5419..5420,1..23)

                     /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)."

ORIGIN

        1 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga

       61 tataccatgg gcagcagcca tcaccatcat caccacagcc aggatccgaa ttcgagctcg

      121 gcgcgcctgc aggtcgacaa gcttgcggcc gcataatgct taagtcgaac agaaagtaat

      181 cgtattgtac acggccgcat aatcgaaatt aatacgactc actatagggg aattgtgagc

      241 ggataacaat tccccatctt agtatattag ttaagtataa gaaggagata tacatatggc

      301 agatctcaat tggatatcgg ccggccacgc gatcgctgac gtcggtaccc tcgagtctgg

      361 taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta ctagcgcagc

      421 ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc

      481 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg

      541 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc

      601 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc

      661 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt

      721 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg

      781 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt

      841 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta

      901 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt

      961 aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat ggcatgagat

     1021 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct

     1081 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta

     1141 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa

     1201 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac

     1261 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa

     1321 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag

     1381 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg

     1441 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag

     1501 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg

     1561 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc

     1621 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat

     1681 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata

     1741 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa

     1801 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca

     1861 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc

     1921 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc

     1981 tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg atacatattt

     2041 gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac gtgagttttc

     2101 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt

     2161 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt

     2221 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat

     2281 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc

     2341 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa

     2401 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg

     2461 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag

     2521 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag

     2581 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa

     2641 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt

     2701 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg

     2761 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc

     2821 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac

     2881 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct

     2941 tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct

     3001 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg

     3061 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat

     3121 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt

     3181 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt

     3241 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg

     3301 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg

     3361 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga

     3421 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta

     3481 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc

     3541 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga

     3601 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga

     3661 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc

     3721 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta

     3781 gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc caccggaagg

     3841 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga

     3901 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt

     3961 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc

     4021 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg

     4081 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt

     4141 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact

     4201 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc

     4261 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc

     4321 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga

     4381 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa

     4441 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg

     4501 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag

     4561 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg

     4621 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc

     4681 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc

     4741 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga

     4801 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg

     4861 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa

     4921 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct

     4981 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg

     5041 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg

     5101 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag

     5161 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac

     5221 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg

     5281 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt

     5341 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaatt

     5401 aatacgactc actatagggg

//