Basic Vector Information
pVP65K 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 ℃.
pVP65K vector Sequence
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"
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