pMCSG35B vector (V010671)

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LOCUS       pMCSG35B.        7484 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding N-terminal 10xHis-MBP-TEV and C-terminal 
            TEV-6xHis plus TVMV protease. See also pMCSG35.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG35B
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7484)
  AUTHORS   Eschenfeldt WH, Maltseva N, Stols L, Donnelly MI, Gu M, Nocek B, Tan
            K, Kim Y, Joachimiak A.
  TITLE     Cleavable C-terminal His-tag vectors for structure determination.
  JOURNAL   J. Struct. Funct. Genomics 2010;11:31-9.
  PUBMED    20213425
REFERENCE   2  (bases 1 to 7484)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7484)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. 
            Funct. Genomics"; date: "2010"; volume: "11"; pages: "31-9"
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..7484
                     /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(208..225)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(232..252)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(263..283)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(305..1402)
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             complement(1430..1456)
                     /label=9xHis
                     /note="9xHis affinity tag"
     CDS             complement(1457..1459)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(1467..1489)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1504..1528)
                     /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(1529..1547)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(1778..1864)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(1900..2607)
                     /label=TVMV protease
                     /note="tobacco vein mottling virus NIa protease
                     (Nallamsetty et al., 2004)"
     promoter        complement(2631..2704)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator
                     sites (Lutz and Bujard, 1997)"
     promoter        2887..2964
                     /label=lacI promoter
     CDS             2965..4044
                     /label=lacI
                     /note="lac repressor"
     protein_bind    4060..4081
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             4856..5044
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    5149..5291
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5477..6065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6239..7096)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7097..7200)
                     /label=AmpR promoter