pRSFDuet-1 Plasmid

pRSFDuet-1

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pRSFDuet-1,Plasmid pRSFDuet-1,pRSFDuet-1 vector

pRSFDuet-1 Informaiton

Promoter: T7/Lac

Replicon: RSF ori

Terminator: T7 Terminator

Plasmid classification: large intestine plasmids, large intestine other plasmids, large intestine double frame plasmid.

Plasmid size: 3829bp

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

Prokaryotic resistance: kanamycin Kan (50 g/ml)

Cloning strains: E. coli DH5 and E.

Culture conditions: 37 C, aerobic, LB

Expression host: E. coli BL21 (DE3).

Culture conditions: 37 C, aerobic, LB

Induction: IPTG or lactose and its analogues.

5'sequencing primers: ACYCDuetUP1-F (GGATCTCGACGCTCTCCCT) DuetUP2-F (TTGTACACGGCCGCATAATC)

3'sequencing primers: DuetDOWN1-R (GATTATGCGGCCGTGTACAA) T7-ter-R (TGCTAGTTATTGCTCAGCGG)

Remarks: simultaneous expression of two foreign proteins

pRSFDuet-1 Description
The pRSFDuet-1 plasmid is a prokaryotic expression vector with a 6*His tag on the N-terminal and a S tag on the C-terminal. The plasmid contains several commonly used restriction sites to facilitate cloning of different genes. The expression was induced by T7 RNA polymerase from host cells. The target gene was cloned into plasmid vector and controlled by strong phage transcription and translation signals.

The plasmid also carries RSF replication factor, lacI gene and chloramphenicol resistant gene. The gene that allows the pETDuet-1 vector to insert the coexpression 4 target gene into MCS 1 in an appropriate host strain can be sequenced using ACYCDuetUP1 and DuetDOWN1 primers. The genes inserted into MCS2 can be sorted by DuetUP2 primers and T7.

pRSFDuet-1 is designed for the coexpression of two target ORFs. The vector contains two multiple cloning sites (MCS), each of which is preceded by a T7lac promoter and ribosome binding site (rbs). The vector also carries the RSF1030-derived RSF replicon, lacI gene and kanamycin resistance gene (KnR). This vector can be used in combination with pACYCDuet?-1 , pCDFDuet?-1 , and pETDuet?-1 in an appropriate host strain for the coexpression of 4 to 8 target proteins. ORFs inserted into MCS1 can be sequenced using the ACYCDuetUP1 Primer and DuetDOWN1 Primer . ORFs inserted into MCS2 can be sequenced using the DuetUP2 Primer and T7 Terminator Primer . Unique sites are shown on the circle map.

pRSFDuet-1 Multiple cloning site

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pRSFDuet-1 Sequence

LOCUS       Exported                3829 bp ds-DNA     circular SYN 26-8-2015
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled 4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3829)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 2015-8-26  
FEATURES             Location/Qualifiers
     source          1..3829
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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)."
     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"
     CDS             complement(739..1554)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin in bacteria or G418
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(1555..1646)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      complement(1662..2411)
                     /direction=LEFT
                     /note="RSF ori"
                     /note="Plasmids containing the RSF 1030 origin of
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(2607..3689)
                     /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(3690..3767)
                     /gene="lacI"
                     /note="lacI promoter"
     promoter        3813..2
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       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 tgaaacctca ggcatttgag aagcacacgg
      541 tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt
      601 aacgaccctg ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt
      661 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      721 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
      781 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
      841 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
      901 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
      961 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     1021 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     1081 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca
     1141 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1201 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1261 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1321 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1381 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1441 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1501 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt
     1561 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     1621 tgaatgtatt tagaaaaata aacaaatagg catgcagcgc tcttccgctt cctcgctcac
     1681 tgactcgcta cgctcggtcg ttcgactgcg gcgagcggtg tcagctcact caaaagcggt
     1741 aatacggtta tccacagaat caggggataa agccggaaag aacatgtgag caaaaagcaa
     1801 agcaccggaa gaagccaacg ccgcaggcgt ttttccatag gctccgcccc cctgacgagc
     1861 atcacaaaaa tcgacgctca agccagaggt ggcgaaaccc gacaggacta taaagatacc
     1921 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     1981 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgtt
     2041 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2101 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2161 acgacttatc gccactggca gcagccattg gtaactgatt tagaggactt tgtcttgaag
     2221 ttatgcacct gttaaggcta aactgaaaga acagattttg gtgagtgcgg tcctccaacc
     2281 cacttacctt ggttcaaaga gttggtagct cagcgaacct tgagaaaacc accgttggta
     2341 gcggtggttt ttctttattt atgagatgat gaatcaatcg gtctatcaag tcaacgaaca
     2401 gctattccgt tactctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     2461 agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg
     2521 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     2581 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2641 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     2701 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     2761 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     2821 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     2881 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     2941 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     3001 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     3061 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     3121 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     3181 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     3241 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     3301 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     3361 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     3421 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     3481 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     3541 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     3601 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     3661 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     3721 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     3781 ctctccctta tgcgactcct gcattaggaa attaatacga ctcactata
//