pMCSG25 vector (V010695)

LOCUS       pMCSG25.        5664 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial expression vector encoding an aspartic endopeptidase fused
            to a 6xHis-TEV leader.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG25
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5664)
  AUTHORS   Osipiuk J, Mulligan R, Bargassa M, Hamilton JE, Cunningham MA, 
            Joachimiak A.
  TITLE     Characterization of member of DUF1888 protein family, self-cleaving 
            and self-assembling endopeptidase.
  JOURNAL   J. Biol. Chem. 2012;287:19452-61.
  PUBMED    22493430
REFERENCE   2  (bases 1 to 5664)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5664)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Biol. 
            Chem."; date: "2012"; volume: "287"; pages: "19452-61"
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..5664
                     /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(223..243)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(268..285)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(292..666)
                     /codon_start=1
                     /product="self-cleaving aspartic endopeptidase SO1698 from 
                     Shewanella oneidensis"
                     /note="aspartic endopeptidase"
                     /translation="MFAPQGLAQFIKVNVTLENGEPVFIYTDANGQVCQGDITVTQAGT
                     ITYLLNDQTLKGLKFVGVGFVTPFDGIIDAVTISSDGMLVQLVDLDKTPGTTKFQFVLS
                     NTANTLLVLSPDPQIINRPQN"
     RBS             complement(674..696)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(711..735)
                     /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(736..754)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1067..1144
                     /label=lacI promoter
     CDS             1145..2224
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2240..2261
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3036..3224
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3329..3471
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3657..4245)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4419..5276)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5277..5380)
                     /label=AmpR promoter