pACYCDuet-1大肠双表达质粒

基本信息

启动子:	T7/Lac
复制子:	p15A 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:	4008bp
质粒标签:	N-6×His,C-S
原核抗性:	氯霉素Chl（50μg/ml）
克隆菌株:	DH5α等大肠杆菌
培养条件:	37℃，有氧，LB
表达宿主:	BL21(DE3)等大肠杆菌
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

         pACYCDuet-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p15A复制因子、lacI基因和氯霉素抗性基因。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。
        pACYCDuet-1 is designed for the coexpression of two target genes. The vector contains two multiple cloning sites (MCS), each of which is preceded by a T7 promoter/lac operator and ribosome binding site (rbs). The vector also carries the P15A replicon, lacI gene and chloramphenicol resistance gene. This vector can be used in combination with pETDuet™-1 in an appropriate host strain for the coexpression of up to 4 target genes. Genes inserted into MCS1 can be sequenced using the ACYCDuetUP1 Primer and DuetDOWN1 Primer . Genes inserted into MCS2 can be sequenced using the DuetUP2 Primer  and T7 Terminator Primer .

质粒图谱

质粒序列

LOCUS       Exported                4008 bp ds-DNA     circular SYN 27-8-2015
DEFINITION  synthetic circular DNA
KEYWORDS    Untitled
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4008)
  TITLE     Direct Submission
  JOURNAL   Exported 2015-8-27   from Plasmid
FEATURES             Location/Qualifiers
     source          1..4008
                     /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(729..1388)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /note="CmR"
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(1389..1491)
                     /note="cat promoter"
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(2017..2561)
                     /direction=LEFT
                     /note="p15A ori"
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells
                     that contain a second plasmid with the ColE1 origin."
     CDS             complement(2786..3868)
                     /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(3869..3946)
                     /gene="lacI"
                     /note="lacI promoter"
     promoter        3992..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 cgaatttgct ttcgaatttc tgccattcat
      661 ccgcttatta tcacttattc aggcgtagca ccaggcgttt aagggcacca ataactgcct
      721 taaaaaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt
      781 ctgccgacat ggaagccatc acagacggca tgatgaacct gaatcgccag cggcatcagc
      841 accttgtcgc cttgcgtata atatttgccc atagtgaaaa cgggggcgaa gaagttgtcc
      901 atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa
      961 aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca
     1021 tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat
     1081 gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc
     1141 accagctcac cgtctttcat tgccatacgg aactccggat gagcattcat caggcgggca
     1201 agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag
     1261 gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc
     1321 tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt
     1381 ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt
     1441 agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat
     1501 tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat
     1561 tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct
     1621 gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg cttctgtttc
     1681 tatcagctgt ccctcctgtt cagctactga cggggtggtg cgtaacggca aaagcaccgc
     1741 cggacatcag cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc
     1801 agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt
     1861 gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
     1921 ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac
     1981 ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga
     2041 caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag
     2101 ataccaggcg tttcccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt
     2161 accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc
     2221 cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg
     2281 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc
     2341 actggcagca gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt
     2401 aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt
     2461 tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt
     2521 tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc
     2581 agataaaata tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca
     2641 gccccatacg atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga
     2701 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     2761 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2821 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     2881 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     2941 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     3001 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     3061 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     3121 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     3181 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     3241 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     3301 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     3361 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     3421 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     3481 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     3541 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     3601 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     3661 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     3721 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     3781 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     3841 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     3901 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     3961 tctcccttat gcgactcctg cattaggaaa ttaatacgac tcactata