pMCSG65 vector (V010617)

LOCUS       pMCSG65.        5369 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for expressing a protein with a 6xHis-HA-TEV leader
            plus a second untagged protein.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG65
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5369)
  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 5369)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5369)
  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     There are two sites for ligation-independent cloning (LIC).
            To express a 6xHis-HA-TEV-tagged protein, linearize with SspI and 
            treat with T4 DNA polymerase plus dGTP.
            To express a second untagged protein, linearize with SmaI and treat 
            with T4 DNA polymerase plus dATP.
FEATURES             Location/Qualifiers
     source          1..5369
                     /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"
     RBS             243..248
                     /note="ribosome binding site"
     CDS             complement(279..299)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(306..332)
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             complement(351..368)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(369..371)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(379..401)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(416..440)
                     /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(441..459)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        772..849
                     /label=lacI promoter
     CDS             850..1929
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1945..1966
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2741..2929
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3034..3176
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3362..3950)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4124..4981)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4982..5085)
                     /label=AmpR promoter