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Plasmid Files

pVP65K

Bacterial Flexi® Vector with a kanamycin resistance marker and the barnase gene, encoding an N-terminal MBP-TVMV-8xHis cassette plus TVMV protease.

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pVP65K Sequence and MappVP65K.dna
Map and Sequence File   
Sequence Author:  Center for Eukaryotic Structural Genomics
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 FspI (6775) NaeI (6375) NgoMIV (6373) RsrII (6358) AvrII (6206) BspHI (6105) PciI (5385) NdeI (5208) BstZ17I (5158) BstEII (4352) ApaI (4331) PspOMI (4327) EcoRV (4088) HpaI (4032) HindIII (3589) BstBI (3586) BspDI - ClaI (3575) SgrDI (3562) BsiWI (1) SacII (267) SpeI (380) BglI (397) DraIII (456) AgeI (617) PaeR7I - XhoI (947) AanI (995) MfeI (1005) RBS NcoI (1059) BsaI (1127) AanI (1416) BmgBI (1606) AfeI (1974) PacI (2169) TVMV site NsiI (2236) AsiSI - SgfI (2244) NotI (2247) Bpu10I (2698) PasI (3157) SspI (3237) ScaI (3342) PmeI (3376) Eco53kI (3389) SacI (3391) SbfI (3424) BsrGI (3530) AscI (3547) pVP65K 7174 bp
FspI  (6775)
1 site
T G C G C A A C G C G T
NaeI  (6375)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
NgoMIV  (6373)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
RsrII  (6358)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
AvrII  (6206)
1 site
C C T A G G G G A T C C
BspHI  (6105)
1 site
T C A T G A A G T A C T
PciI  (5385)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NdeI  (5208)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
BstZ17I  (5158)
1 site
G T A T A C C A T A T G
BstEII  (4352)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
ApaI  (4331)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (4327)
1 site
G G G C C C C C C G G G
EcoRV  (4088)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
HpaI  (4032)
1 site
G T T A A C C A A T T G
HindIII  (3589)
1 site
A A G C T T T T C G A A
BstBI  (3586)
1 site
T T C G A A A A G C T T
BspDI  (3575)
1 site
A T C G A T T A G C T A
ClaI  (3575)
1 site
A T C G A T T A G C T A
SgrDI  (3562)
1 site
C G T C G A C G G C A G C T G C
BsiWI  (1)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
SacII  (267)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
SpeI  (380)
1 site
A C T A G T T G A T C A
BglI  (397)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
DraIII  (456)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
AgeI  (617)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
PaeR7I  (947)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (947)
1 site
C T C G A G G A G C T C
AanI  (995)
2 sites
T T A T A A A A T A T T
MfeI  (1005)
1 site
C A A T T G G T T A A C
NcoI  (1059)
1 site
C C A T G G G G T A C C
BsaI  (1127)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
AanI  (1416)
2 sites
T T A T A A A A T A T T
BmgBI  (1606)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
AfeI  (1974)
1 site
A G C G C T T C G C G A
PacI  (2169)
1 site
T T A A T T A A A A T T A A T T
NsiI  (2236)
1 site
A T G C A T T A C G T A
AsiSI  (2244)
1 site
G C G A T C G C C G C T A G C G
SgfI  (2244)
1 site
G C G A T C G C C G C T A G C G
NotI  (2247)
1 site
G C G G C C G C C G C C G G C G
Bpu10I  (2698)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends
generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
PasI  (3157)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
SspI  (3237)
1 site
A A T A T T T T A T A A
ScaI  (3342)
1 site
A G T A C T T C A T G A
PmeI  (3376)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (3389)
1 site
G A G C T C C T C G A G
SacI  (3391)
1 site
G A G C T C C T C G A G
SbfI  (3424)
1 site
C C T G C A G G G G A C G T C C
BsrGI  (3530)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
AscI  (3547)
1 site
G G C G C G C C C C G C G C G G
MBP
1061 .. 2164  =  1104 bp
368 amino acids  =  40.4 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal
sequence, so MBP will remain in the cytosol.
MBP
1061 .. 2164  =  1104 bp
368 amino acids  =  40.4 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal
sequence, so MBP will remain in the cytosol.
lacI
3807 .. 4889  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
3807 .. 4889  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
NeoR/KanR
6212 .. 7006  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
6212 .. 7006  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
TVMV protease
139 .. 849  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease
(Nallamsetty et al., 2004)
TVMV protease
139 .. 849  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease
(Nallamsetty et al., 2004)
CmR
2712 .. 3371  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
2712 .. 3371  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
5446 .. 6034  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5446 .. 6034  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
barnase
2277 .. 2612  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
barnase
2277 .. 2612  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
rrnB T1 terminator
20 .. 106  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
20 .. 106  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
3628 .. 3714  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
3628 .. 3714  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
lacI promoter
4890 .. 4967  =  78 bp
lacI promoter
4890 .. 4967  =  78 bp
PLtetO-1 promoter
873 .. 946  =  74 bp
   Segment 5:  
   873 .. 898  =  26 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
873 .. 946  =  74 bp
   Segment 4:  -10  
   899 .. 904  =  6 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
873 .. 946  =  74 bp
   Segment 3:  
   905 .. 921  =  17 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
873 .. 946  =  74 bp
   Segment 2:  -35  
   922 .. 927  =  6 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
873 .. 946  =  74 bp
   Segment 1:  
   928 .. 946  =  19 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
873 .. 946  =  74 bp
5 segments
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
T5 promoter
956 .. 1000  =  45 bp
   Segment 1:  
   956 .. 970  =  15 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
956 .. 1000  =  45 bp
   Segment 2:  -35  
   971 .. 976  =  6 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
956 .. 1000  =  45 bp
   Segment 3:  
   977 .. 993  =  17 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
956 .. 1000  =  45 bp
   Segment 4:  -10  
   994 .. 1000  =  7 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
956 .. 1000  =  45 bp
4 segments
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
lac UV5 promoter
2628 .. 2658  =  31 bp
   Segment 1:  -35  
   2628 .. 2633  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2628 .. 2658  =  31 bp
   Segment 2:  
   2634 .. 2651  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2628 .. 2658  =  31 bp
   Segment 3:  -10  
   2652 .. 2658  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2628 .. 2658  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
8xHis
2210 .. 2233  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
2210 .. 2233  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
TVMV site
2189 .. 2209  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa
protease recognition and cleavage site
TVMV site
2189 .. 2209  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa
protease recognition and cleavage site
lac operator
1008 .. 1024  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
1008 .. 1024  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
RBS
1047 .. 1052  =  6 bp
ribosome binding site
RBS
1047 .. 1052  =  6 bp
ribosome binding site
tet operator
903 .. 921  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
903 .. 921  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
928 .. 946  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
928 .. 946  =  19 bp
bacterial operator O2 for the tetR and tetA genes
lac operator
976 .. 992  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
976 .. 992  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
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