pCDFDuet-1大肠双表达质粒

基本信息

启动子:	T7/Lac
复制子:	CloDF13 ori
终止子:	T7 terminator
质粒分类:	大肠系列质粒；大肠其它质粒；大肠双框质粒
质粒大小:	3781bp
质粒标签:	N-6×His,C-S
原核抗性:	链霉素Str（50μg/ml）
克隆菌株:	大肠杆菌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:GATTATGCGGCCGTGTACAA T7-ter:TGCTAGTTATTGCTCAGCGG
备注:	同时表达两个外源蛋白

质粒简介

         pCDFDuet-1质粒是一种原核表达载体， N端含有一个6×His标签，C端含有一个S标签。该质粒含有多个常用的酶切位点，便于不同基因克隆。表达由宿主细胞提供的T7 RNA聚合酶诱导，目的基因被克隆到质粒载体上，受噬菌体强转录及翻译信号控制。该质粒还携带有CloDF13复制因子、lacI基因和链霉素抗性基因。可以使这个pETDuet-1载体在一个适当的宿主菌株的coexpression 4目标基因插入MCS1的基因可以使用ACYCDuetUP1引物和DuetDOWN1引物进行测序。插入MCS2的基因可以用DuetUP2引物和T7进行排序。
        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.

质粒图谱

质粒序列

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  from MLCC
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