pVP33K vector (V010570)

LOCUS       pVP33K.        7253 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial Flexi(R) Vector with a kanamycin resistance marker, for 
            appending an N-terminal 8xHis-MBP-HRV 3C-tetracysteine-TEV cassette 
            to an expressed protein.
ACCESSION   .
VERSION     .
KEYWORDS    pVP33K
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7253)
  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 7253)
  AUTHORS   Center for Eukaryotic Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7253)
  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..7253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        126..170
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA
                     polymerase, with embedded lac operator"
     protein_bind    178..194
                     /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             217..222
                     /note="ribosome binding site"
     CDS             231..233
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             237..260
                     /label=8xHis
                     /note="8xHis affinity tag"
     CDS             267..1364
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             1374..1397
                     /label=HRV 3C site
                     /note="recognition and cleavage site for human rhinovirus
                     3C and PreScission proteases"
     CDS             1398..1415
                     /label=tetracysteine tag
                     /note="tetracysteine peptide that binds biarsenical
                     labeling reagents"
     CDS             1416..1436
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             1465..1797
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
     promoter        1816..1846
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1900..2556
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     misc_feature    2569..2624
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     protein_bind    complement(2712..2732)
                     /label=attB2
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     terminator      2801..2895
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2939..3595
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     terminator      3663..3749
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    complement(3808..3829)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3845..4924)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4925..5002)
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     misc_feature    5194..5334
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5520..6108)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6288..7079)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"