pMCSG75 vector (V010599)

LOCUS       pMCSG75.        5876 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding tRNA genes for rare Arg and Ile codons, 
            plus a C-terminal TEV-AviTag(TM)-6xHis cassette, plus the BirA 
            biotin ligase.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG75
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5876)
  AUTHORS   Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI, 
            Jedrzejczak R, Joachimiak A.
  TITLE     New LIC vectors for production of proteins from genes containing 
            rare codons.
  JOURNAL   J. Struct. Funct. Genomics 2013;14:135-44.
  PUBMED    24057978
REFERENCE   2  (bases 1 to 5876)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5876)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. 
            Funct. Genomics"; date: "2013"; volume: "14"; pages: "135-44"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SmaI and 
            treat with T4 DNA polymerase plus dATP.
FEATURES             Location/Qualifiers
     source          1..5876
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(244..1206)
                     /label=BirA
                     /note="E. coli biotin protein ligase"
     RBS             1215..1220
                     /note="ribosome binding site"
     CDS             complement(1258..1275)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(1282..1326)
                     /label=AviTag(TM)
                     /note="peptide tag that allows for enzymatic biotinylation"
     CDS             complement(1333..1353)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     RBS             complement(1365..1385)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1401..1425)
                     /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)."
     promoter        complement(1426..1444)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(1719..1795)
                     /label=argU
     tRNA            complement(2065..2140)
                     /label=ileX
     promoter        2280..2357
                     /label=lacI promoter
     CDS             2358..3437
                     /label=lacI
                     /note="lac repressor"
     protein_bind    3453..3474
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3869..4457)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4631..5488)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5489..5592)
                     /label=AmpR promoter