pCOLADuet-1大肠双表达质粒

基本信息

启动子:	T7/Lac
复制子:	ColA ori
终止子:	T7 terminator
质粒分类:	大肠杆菌载体；双表达框质粒
质粒大小:	3719bp
质粒标签:	N-6×His,C-S
原核抗性:	卡那青霉素Kan
克隆菌株:	DH5α
培养条件:	37℃，有氧，LB
表达宿主:	BL21(DE3)
培养条件:	37℃，有氧，LB
诱导方式:	IPTG或乳糖及其类似物
5'测序引物:	ACYCDuetUP1：GGATCTCGACGCTCTCCCT DuetUP2:TTGTACACGGCCGCATAATC
3'测序引物:	DuetDOWN1:GATTAGCGGCCGTGTACAA T7-ter:TGCTAGTTATTGCTCAGCGG
备注:	同时表达两个外源蛋白

质粒简介

pCOLADuet-1 is designed for the coexpression of two target genes from a single plasmid. The vector encodes two multiple cloning sites (MCS) each of which is preceded by a T7 promoter, lac operator, and ribosome binding site (rbs). MCS-1 encodes the six-amino acid His•Tag sequence for the creation of a N-terminal fusion and MCS2 encodes the 15 amino acid S•Tag™ peptide after the last restriction site for the creation of a C-terminal fusion if desired. Genes inserted into MCS-1 can be sequenced using the ACYCDuetUP1 Primer and DuetDOWN1 Primer. Genes inserted into MCS-2 can be sequenced using the DuetUP2 Primer and T7 Terminator Primer. The vector has the COLA replicon from ColA(1) and the kanamycin resistance gene. This vector can be transformed into the same cell with plasmids containing compatible origins of replication and drug resistance genes for coexpression of up to 8 target genes. 

质粒图谱

质粒序列

LOCUS       Exported                3719 bp ds-DNA    circular SYN 26-8-2015
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    Untitled
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3719)
  AUTHORS   admin
  TITLE     Direct Submission
  JOURNAL   Exported 2015-8-26  from MLCC
FEATURES             Location/Qualifiers
     source          1..3719
                     /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(1664..2299)
                     /direction=LEFT
                     /note="ColA ori"
                     /note="Plasmids containing the ColA origin of replication
                     can be propagated in E. coli cells that contain additional
                     plasmids with compatible origins."
     CDS             complement(2497..3579)
                     /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(3580..3657)
                     /gene="lacI"
                     /note="lacI promoter"
                     /note="
                     "
     promoter        3703..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 catgctagcg cagaaacgtc ctagaagatg
     1681 ccaggaggat acttagcaga gagacaataa ggccggagcg aagccgtttt tccataggct
     1741 ccgcccccct gacgaacatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac
     1801 aggactataa agataccagg cgtttccccc tgatggctcc ctcttgcgct ctcctgttcc
     1861 cgtcctgcgg cgtccgtgtt gtggtggagg ctttacccaa atcaccacgt cccgttccgt
     1921 gtagacagtt cgctccaagc tgggctgtgt gcaagaaccc cccgttcagc ccgactgctg
     1981 cgccttatcc ggtaactatc atcttgagtc caacccggaa agacacgaca aaacgccact
     2041 ggcagcagcc attggtaact gagaattagt ggatttagat atcgagagtc ttgaagtggt
     2101 ggcctaacag aggctacact gaaaggacag tatttggtat ctgcgctcca ctaaagccag
     2161 ttaccaggtt aagcagttcc ccaactgact taaccttcga tcaaaccgcc tccccaggcg
     2221 gttttttcgt ttacagagca ggagattacg acgatcgtaa aaggatctca agaagatcct
     2281 ttacggattc ccgacaccat cactctagat ttcagtgcaa tttatctctt caaatgtagc
     2341 acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc
     2401 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     2461 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2521 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2581 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     2641 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     2701 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     2761 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     2821 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     2881 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     2941 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     3001 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     3061 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     3121 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     3181 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     3241 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     3301 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     3361 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     3421 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     3481 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     3541 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     3601 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     3661 gatctcgacg ctctccctta tgcgactcct gcattaggaa attaatacga ctcactata