pCDFDuet-1
Search name
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 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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