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載體描述

pACYCDue-1 is designed for the co-expression 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 pACYCDuet?-1 (Cat. No. 71146-3) 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 (Cat. No. 71178-3) and DuetDOWN1 Primer (Cat. No. 71179- 3). Genes inserted into MCS2 can be sequenced using the DuetUP2 Primer (Cat. No. 71180-3) and T7 Terminator Primer (Cat. No. 69337-3).

載體序列

LOCUS       Exported                4008 bp ds-DNA     circular SYN 23-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    pACYCDuet-1

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 4008)

  AUTHORS   BT-Lab

  TITLE     Direct Submission

  JOURNAL   Exported Saturday, Mar 23, 2019 from SnapGene Viewer 4.3.4

FEATURES             Location/Qualifiers

     source          1..4008

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     protein_bind    3..27

                     /label=lac operator

                     /bound_moiety="lac repressor encoded by 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)."

     CDS             83..100

                     /codon_start=1

                     /product="6xHis affinity tag"

                     /label=6xHis

                     /translation="HHHHHH"

     misc_feature    106..195

                     /label=MCS1

                     /note="multiple cloning site"

     primer_bind     214..233

                     /label=T7 primer

     promoter        214..232

                     /label=T7 promoter

                     /note="promoter for bacteriophage T7 RNA polymerase"

     protein_bind    233..257

                     /label=lac operator

                     /bound_moiety="lac repressor encoded by 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)."

     misc_feature    297..359

                     /label=MCS2

                     /note="multiple cloning site"

     CDS             366..410

                     /codon_start=1

                     /product="affinity and epitope tag derived from pancreatic

                     ribonuclease A"

                     /label=S-Tag

                     /translation="KETAAAKFERQHMDS"

     primer_bind     complement(448..467)

                     /label=T7 ter primer

     terminator      462..509

                     /label=T7 terminator

                     /note="transcription terminator for bacteriophage T7 RNA

                     polymerase"

     CDS             complement(729..1388)

                     /codon_start=1

                     /gene="cat"

                     /product="chloramphenicol acetyltransferase"

                     /label=CmR

                     /note="confers resistance to chloramphenicol"

                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL

                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS

                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM

                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"

     promoter        complement(1389..1491)

                     /label=cat promoter

                     /note="promoter of the E. coli cat gene encoding

                     chloramphenicol acetyltransferase"

     rep_origin      complement(2017..2561)

                     /direction=LEFT

                     /label=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"

                     /label=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"

                     /label=lacI promoter

     primer_bind     join(3992..4008,1..3)

                     /label=T7 primer

     promoter        join(3992..4008,1..2)

                     /label=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

//