Basic Vector Information
pVP33K vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
pVP33K vector Sequence
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"
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