pMCSG14 vector (V010717)

LOCUS       pMCSG14.        4754 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial coexpression vector with a 6xHis-GST-TEV leader and a p15A
            origin, for high-throughput purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG14
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4754)
  AUTHORS   Eschenfeldt WH, Lucy S, Millard CS, Joachimiak A, Mark ID.
  TITLE     A family of LIC vectors for high-throughput cloning and purification
            of proteins.
  JOURNAL   Methods Mol. Biol. 2009;498:105-15.
  PUBMED    18988021
REFERENCE   2  (bases 1 to 4754)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4754)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods 
            Mol. Biol."; date: "2009"; volume: "498"; pages: "105-15"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SspI and 
            treat with T4 DNA polymerase plus dGTP.
FEATURES             Location/Qualifiers
     source          1..4754
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /label=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)."
     RBS             42..64
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             71..73
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             83..100
                     /label=6xHis
                     /note="6xHis affinity tag"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /label=lac repressor encoded by lacI binding site
                     /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)."
     RBS             286..291
                     /note="ribosome binding site"
     CDS             300..302
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             303..320
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             339..992
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
     CDS             1020..1040
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             1112..1156
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     terminator      1208..1255
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1478..2134)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(2135..2237)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(2763..3307)
                     /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."
     protein_bind    complement(3498..3519)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3535..4614)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4615..4692)
                     /label=lacI promoter