pCDFDuet-1
PVT0104 2ug
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
421 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
481 cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg
541 tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt
601 aacgaccctg ccctgaaccg acgaccgggt catcgtggcc ggatcttgcg gcccctcggc
661 ttgaacgaat tgttagacat tatttgccga ctaccttggt gatctcgcct ttcacgtagt
721 ggacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttct tgtccaagat
781 aagcctgtct agcttcaagt atgacgggct gatactgggc cggcaggcgc tccattgccc
841 agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac caaatgcggg
901 acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag ttccatagcg
961 ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca aagagttcct
1021 ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc aagatagcca
1081 gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg cgctgccatt
1141 ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg tcgtgcacaa
1201 caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc gaagtttcca
1261 aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc accgtaacca
1321 gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg tacaaatgta
1381 cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct gatagttgag
1441 tcgatacttc ggcgatcacc gcttccctca tactcttcct ttttcaatat tattgaagca
1501 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
1561 aaatagctag ctcactcggt cgctacgctc cgggcgtgag actgcggcgg gcgctgcgga
1621 cacatacaaa gttacccaca gattccgtgg ataagcaggg gactaacatg tgaggcaaaa
1681 cagcagggcc gcgccggtgg cgtttttcca taggctccgc cctcctgcca gagttcacat
1741 aaacagacgc ttttccggtg catctgtggg agccgtgagg ctcaaccatg aatctgacag
1801 tacgggcgaa acccgacagg acttaaagat ccccaccgtt tccggcgggt cgctccctct
1861 tgcgctctcc tgttccgacc ctgccgttta ccggatacct gttccgcctt tctcccttac
1921 gggaagtgtg gcgctttctc atagctcaca cactggtatc tcggctcggt gtaggtcgtt
1981 cgctccaagc tgggctgtaa gcaagaactc cccgttcagc ccgactgctg cgccttatcc
2041 ggtaactgtt cacttgagtc caacccggaa aagcacggta aaacgccact ggcagcagcc
2101 attggtaact gggagttcgc agaggatttg tttagctaaa cacgcggttg ctcttgaagt
2161 gtgcgccaaa gtccggctac actggaagga cagatttggt tgctgtgctc tgcgaaagcc
2221 agttaccacg gttaagcagt tccccaactg acttaacctt cgatcaaacc acctccccag
2281 gtggtttttt cgtttacagg gcaaaagatt acgcgcagaa aaaaaggatc tcaagaagat
2341 cctttgatct tttctactga accgctctag atttcagtgc aatttatctc ttcaaatgta
2401 gcacctgaag tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg
2461 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
2521 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
2581 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
2641 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
2701 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
2761 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
2821 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
2881 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
2941 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
3001 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
3061 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
3121 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
3181 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
3241 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
3301 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
3361 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
3421 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
3481 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
3541 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
3601 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
3661 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
3721 gggatctcga cgctctccct tatgcgactc ctgcattagg aaattaatac gactcactat
3781 a
//
Caution:
1. This product is FOR RESEARCH USE ONLY!
2. The item is lyophilized form, Please take the powder plasmid by centrifugation at 5000rpm/min for 1min. Add 20μl ddH2O in to the tube of plasmid.
Search name
pCDFDuet-1,Plasmid pCDFDuet-1,pCDFDuet-1 vector
