pMCSG30 vector (V010685)

LOCUS       pMCSG30.        8314 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial expression vector with a 6xHis-TEV-MBP leader and a SacB 
            negative selection marker, for high-throughput purification of 
            recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG30
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8314)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8314)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SfiI to 
            remove the SacB cassette, and treat with T4 DNA polymerase plus 
            dTTP.
FEATURES             Location/Qualifiers
     source          1..8314
                     /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(258..1676)
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     promoter        complement(1677..2122)
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             complement(2147..3244)
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             complement(3251..3271)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(3296..3313)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(3314..3316)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(3324..3346)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(3361..3385)
                     /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(3386..3404)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3717..3794
                     /label=lacI promoter
     CDS             3795..4874
                     /label=lacI
                     /note="lac repressor"
     protein_bind    4890..4911
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             5686..5874
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    5979..6121
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(6307..6895)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7069..7926)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7927..8030)
                     /label=AmpR promoter