pMCSG62 vector (V010623)

LOCUS       pMCSG62.        5869 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding tRNA genes for rare Arg and Ile codons, 
            for expressing a protein with a 6xHis-AviTag(TM)-TEV leader plus the
            BirA biotin ligase.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG62
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5869)
  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 5869)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5869)
  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 SspI and 
            treat with T4 DNA polymerase plus dGTP.
FEATURES             Location/Qualifiers
     source          1..5869
                     /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(1251..1271)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(1278..1322)
                     /label=AviTag(TM)
                     /note="peptide tag that allows for enzymatic biotinylation"
     CDS             complement(1329..1346)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(1347..1349)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(1357..1379)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1394..1418)
                     /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(1419..1437)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(1712..1788)
                     /label=argU
     tRNA            complement(2058..2133)
                     /label=ileX
     promoter        2273..2350
                     /label=lacI promoter
     CDS             2351..3430
                     /label=lacI
                     /note="lac repressor"
     protein_bind    3446..3467
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3862..4450)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4624..5481)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5482..5585)
                     /label=AmpR promoter