pCDFDuet- 1 Plasmid

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pCDFDuet-1

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pCDFDuet-1,Plasmid pCDFDuet-1,pCDFDuet-1 vector

 

 

pCDFDuet-1 Information

Promoter: T7/Lac

Copier: CloDF13 ori

Terminator: T7 Terminator

Plasmid classification: large intestine series plasmids; other large intestine plasmids; large intestine double-framed plasmids

Plasmid size: 3781bp

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

Prokaryotic resistance: streptomycin Str (50 ug/ml)

Cloning strain: Escherichia coli 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: ACYCDuetUP1: GGATCTCGACGCTCCCT DuetUP2: TTGTACACGCCGCATAATC

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

Note: Expressing two exogenous proteins at the same time

 

pCDFDuet-1 Description

The pCDFDuet-1 plasmid is a prokaryotic expression vector. The N-terminal contains a 6*His tag and the C-terminal contains an S-tag. The plasmid contains several commonly used restriction sites, which facilitates the cloning of different genes. The expression was induced by T7 RNA polymerase provided by host cells. The target gene was cloned into plasmid vector and controlled by strong transcription and translation signals of bacteriophages. The plasmid also carries CloDF13 replication factor, lacI gene and streptomycin resistance gene. The coexpression 4 target gene of the pETDuet-1 vector can be inserted into the gene of MCS 1 in an appropriate host strain. ACYCDuetUP1 primer and DuetDOWN1 primer can be used for sequencing. The insertion of MCS2 gene can be sequenced by DuetUP2 primer and T7.

 

pCDFDuet-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 CloDF13-derived CDF replicon, lacI gene and streptomycin/spectinomycin resistance gene (SmR). This vector can be used in combination with pACYCDuet™-1 , pRSFDuet™-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.

 

pCDFDuet-1 Multiple cloning site

pCDFDuet-1-vector

 

 

 

pCDFDuet-1 Sequence

LOCUS       Exported                3781 bp ds-DNA     circular SYN 26-8-2015
DEFINITION  synthetic circular DNA
KEYWORDS    Untitled
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3781)
  TITLE     Direct Submission
  JOURNAL   Exported 2015-8-26   
FEATURES             Location/Qualifiers
     source          1..3781
                     /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(680..1471)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy,
                     1985)"
                     /note="SmR"
                     /note="confers resistance to spectinomycin and
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(1472..1563)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      complement(1611..2349)
                     /direction=LEFT
                     /note="CloDF13 ori"
                     /note="Plasmids containing the CloDF13 (CDF) origin of
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(2559..3641)
                     /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(3642..3719)
                     /gene="lacI"
                     /note="lacI promoter"
     promoter        3765..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
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