pETDuet-1

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  • Model: PVT0019
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pETDuet-1

PVT0019 2ug

 

pETDuet-1 Information

Promoter: T7/Lac

Copier: ColE1 ori, F1ori

Terminator: T7 Terminator

Plasmid Classification: Escherichia coli Vector; Double Expression Frame Plasmid

Plasmid size: 5420 BP

Plasmid label: N-6 *His, C-S

Prokaryotic resistance: ampicillin Amp

Cloning strain: DH5alpha

Culture conditions: 37 C, aerobic, LB

Expression host: BL21 (DE3)

Culture conditions: 37 C, aerobic, LB

Induction: IPTG or lactose and its analogues

5'Sequencing primers: pET Upstream: ATGTCCGGCGTAGA DuetUP2: TTGTACACGCCGCATAATC

3'Sequencing primers: DuetDOWN1: GATTAGCGCCGTGTACAA T7-ter: TGCTAGTTATTGCTCAGCGG

Note: Expressing two exogenous proteins at the same time

 

pETDuet-1 Description

pETDuet-1 is designed to express two exogenous genes simultaneously in E. coli. The vector contains two polyclonal loci, each of which is composed of a T7 promoter/Lac operator and a ribosome binding site. PETDuet-1 carries ColE1 replicon, lacI gene and ampicillin resistance gene from pBR322 plasmid. The vector can be used in conjunction with pACYCDuet-1 vector. In suitable host bacteria, it can express up to four target genes together. The target gene inserted in polyclonal site 1 of pETDuet-1 vector can be sequenced by pET Upstream primer and DuetDOWN1 primer. The target gene inserted in polyclonal site 2 can be sequenced by DuetUP2 primer and T7 Terminator primer.

 

pETDuet-1 Cloning and expression regions

pETDuet-1-cloning-expression-regions

pETDuet-1 Sequence

LOCUS       Exported                5420 bp ds-DNA     circular SYN 13-8-2015
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5420)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 2015-8-13  
FEATURES             Location/Qualifiers
     source          1..5420
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          42..64
                     /organism="Enterobacteria phage T7"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:10760"
     protein_bind    3..27
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     promoter        214..232
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /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)."
     CDS             366..410
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic
                     ribonuclease A"
                     /note="S-Tag"
                     /translation="KETAAAKFERQHMDS"
     terminator      462..509
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     rep_origin      546..1001
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(1116..1976)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(1977..2069)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      2150..2738
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     misc_feature    2924..3066
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     CDS             complement(3168..3359)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3931..5013)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="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(5014..5091)
                     /gene="lacI"
                     /note="lacI promoter"
     promoter        5404..2
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tggcagatct caattggata tcggccggcc acgcgatcgc tgacgtcggt accctcgagt
      361 ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg
      421 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
      481 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
      541 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
      601 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
      661 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
      721 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
      781 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
      841 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
      901 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
      961 atttaacgcg aattttaaca aaatattaac gtttacaatt tctggcggca cgatggcatg
     1021 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     1081 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     1141 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     1201 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     1261 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1321 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1381 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     1441 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     1501 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1561 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     1621 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     1681 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     1741 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     1801 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     1861 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     1921 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     1981 ttcctttttc aatcatgatt gaagcattta tcagggttat tgtctcatga gcggatacat
     2041 atttgaatgt atttagaaaa ataaacaaat aggtcatgac caaaatccct taacgtgagt
     2101 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2161 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2221 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2281 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2341 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2401 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2461 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2521 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2581 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2641 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2701 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2761 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2821 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2881 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     2941 tccttacgca tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg
     3001 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3061 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3121 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3181 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3241 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3301 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3361 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3421 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     3481 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     3541 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     3601 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     3661 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     3721 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     3781 cctagccggg tcctcaacga caggagcacg atcatgctag tcatgccccg cgcccaccgg
     3841 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     3901 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3961 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4021 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4081 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4141 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4201 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4261 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4321 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     4381 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4441 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4501 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4561 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4621 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4681 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4741 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4801 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4861 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4921 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     4981 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5041 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5101 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     5161 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     5221 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     5281 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     5341 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga
     5401 aattaatacg actcactata
//

 

*Caution

Product is for research used only!
 

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