pMCSG18 载体 (V010713#)

载体基本信息
载体名称 pMCSG18 载体抗性 Ampicillin
载体长度 6009 bp 载体类型 Structural Genomics Vectors
载体来源 Stols L, Gu M, Dieckman L, Raffen R, Collart FR, Donnelly MI. 拷贝数 High copy number

pMCSG18 载体载体图谱

pMCSG186009 bp30060090012001500180021002400270030003300360039004200450048005100540057006000T7 terminator6xHisGFPTEV site6xHisATGRBSlac operatorT7 promoterlacI promoterlacIroporibla(M)AmpR promoter

pMCSG18 载体载体序列

LOCUS       Exported                6009 bp ds-DNA    circular SYN 05-4-2014
DEFINITION  Bacterial vector with a 6xHis-TEV leader for high-throughput 
            purification of C-terminally GFP-tagged proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG18
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6009)
  AUTHORS   Stols L, Gu M, Dieckman L, Raffen R, Collart FR, Donnelly MI.
  TITLE     A new vector for high-throughput, ligation-independent cloning 
            encoding a tobacco etch virus protease cleavage site.
  JOURNAL   Protein Expr. Purif. 2002;25:8-15.
  PUBMED    12071693
REFERENCE   2  (bases 1 to 6009)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
COMMENT     For ligation-independent cloning (LIC), linearize with SspI and 
            treat with T4 DNA polymerase plus dGTP.
FEATURES             Location/Qualifiers
     source          1..6009
                     /organism="synthetic DNA construct"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
     terminator      26..73
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(208..921)
                     /codon_start=1
                     /product="green fluorescent protein variant"
                     /note="GFP"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLCYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYN"
     CDS             complement(946..966)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /note="TEV site"
                     /translation="ENLYFQS"
     CDS             complement(991..1008)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(1009..1011)
                     /codon_start=1
                     /product="start codon"
                     /note="ATG"
                     /translation="M"
     RBS             1020..1025
                     /note="ribosome binding site"
     protein_bind    1056..1080
                     /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)."
     promoter        complement(1081..1099)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1412..1489
                     /gene="lacI"
                     /note="lacI promoter"
                     /note="
                     "
     CDS             1490..2572
                     /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"
     CDS             3381..3572
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     rep_origin      complement(4002..4590)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4761..5549)
                     /codon_start=1
                     /gene="bla(M)"
                     /product="beta-lactamase lacking the signal sequence"
                     /note="bla(M)"
                     /note="allows cytosolic expression of beta-lactamase"
                     /translation="PETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMST
                     FKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMS
                     DNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATT
                     LRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAA
                     LGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     promoter        complement(5622..5725)
                     /gene="bla"
                     /note="AmpR promoter"

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