pVP65K vector (V010567)

LOCUS       pVP65K.        7174 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial Flexi(R) Vector with a kanamycin resistance marker and the
            barnase gene, encoding an N-terminal MBP-TVMV-8xHis cassette plus 
            TVMV protease.
ACCESSION   .
VERSION     .
KEYWORDS    pVP65K
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7174)
  AUTHORS   Blommel PG, Martin PA, Wrobel RL, Steffen E, Fox BG.
  TITLE     High efficiency single step production of expression plasmids from 
            cDNA clones using the Flexi Vector cloning system.
  JOURNAL   Protein Expr. Purif. 2006;47:562-70.
  PUBMED    16377204
REFERENCE   2  (bases 1 to 7174)
  AUTHORS   Center for Eukaryotic Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7174)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protein 
            Expr. Purif."; date: "2006"; volume: "47"; pages: "562-70"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     To remove the lethal barnase gene, insert an ORF between the SgfI 
            site and the PmeI site or a downstream site.
FEATURES             Location/Qualifiers
     source          1..7174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(20..106)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(142..849)
                     /label=TVMV protease
                     /note="tobacco vein mottling virus NIa protease
                     (Nallamsetty et al., 2004)"
     promoter        complement(873..946)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator
                     sites (Lutz and Bujard, 1997)"
     promoter        956..1000
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA
                     polymerase, with embedded lac operator"
     protein_bind    1008..1024
                     /label=lac repressor encoded by lacI binding site
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="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)."
     RBS             1047..1052
                     /note="ribosome binding site"
     CDS             1067..2164
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             2189..2209
                     /label=TVMV site
                     /note="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site"
     CDS             2210..2233
                     /label=8xHis
                     /note="8xHis affinity tag"
     CDS             2277..2609
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
     promoter        2628..2658
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2712..3368
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     misc_feature    3381..3436
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     protein_bind    complement(3521..3541)
                     /label=attB2
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     terminator      3628..3714
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    complement(3773..3794)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3810..4889)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4890..4967)
                     /label=lacI promoter
     misc_feature    5120..5260
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5446..6034)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6215..7006)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"