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Plasmid Files

pVP81K

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

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pVP81K Sequence and MappVP81K.dna
Map and Sequence File   
Sequence Author:  Center for Eukaryotic Structural Genomics
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 EagI (8331) FspI (8139) NaeI (7739) NgoMIV (7737) RsrII (7722) AvrII (7570) BspHI (7469) NdeI (6572) BstEII (5716) ApaI (5695) PspOMI (5691) BstBI (4950) AatII (4927) SgrDI (4926) ZraI (4925) AscI (4911) SbfI (4788) SmaI (4763) TspMI - XmaI (4761) SacI (4755) Eco53kI (4753) PmeI (4740) ScaI (4706) SspI (4601) PasI (4521) BsiWI (1) SpeI (380) BglI (397) DraIII (456) AgeI (617) PaeR7I - XhoI (947) AanI (995) MfeI (1005) lac operator BsaI (1127) AanI (1416) BmgBI (1606) AfeI (1974) PacI (2169) TVMV site NsiI (2236) AsiSI - SgfI (2244) StuI * (2726) AanI (3125) SnaBI (3234) pVP81K 8538 bp
EagI  (8331)
1 site
C G G C C G G C C G G C
FspI  (8139)
1 site
T G C G C A A C G C G T
NaeI  (7739)
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  (7737)
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  (7722)
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  (7570)
1 site
C C T A G G G G A T C C
BspHI  (7469)
1 site
T C A T G A A G T A C T
NdeI  (6572)
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.
BstEII  (5716)
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  (5695)
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  (5691)
1 site
G G G C C C C C C G G G
BstBI  (4950)
1 site
T T C G A A A A G C T T
AatII  (4927)
1 site
G A C G T C C T G C A G
SgrDI  (4926)
1 site
C G T C G A C G G C A G C T G C
ZraI  (4925)
1 site
G A C G T C C T G C A G
AscI  (4911)
1 site
G G C G C G C C C C G C G C G G
SbfI  (4788)
1 site
C C T G C A G G G G A C G T C C
SmaI  (4763)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (4761)
1 site
C C C G G G G G G C C C
XmaI  (4761)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SacI  (4755)
1 site
G A G C T C C T C G A G
Eco53kI  (4753)
1 site
G A G C T C C T C G A G
PmeI  (4740)
1 site
G T T T A A A C C A A A T T T G
ScaI  (4706)
1 site
A G T A C T T C A T G A
SspI  (4601)
1 site
A A T A T T T T A T A A
PasI  (4521)
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.
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.
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)
3 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
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)
3 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
StuI  (2726)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
AanI  (3125)
3 sites
T T A T A A A A T A T T
SnaBI  (3234)
1 site
T A C G T A A T G C A T
SacB
2485 .. 3906  =  1422 bp
473 amino acids  =  53.0 kDa
   Segment 1:  signal peptide  
   2485 .. 2571  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders
bacterial growth sensitive to sucrose
negative selection marker
SacB
2485 .. 3906  =  1422 bp
473 amino acids  =  53.0 kDa
   Segment 2:  
   2572 .. 3906  =  1335 bp
   444 amino acids  =  50.0 kDa
Product: secreted levansucrase that renders
bacterial growth sensitive to sucrose
negative selection marker
SacB
2485 .. 3906  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
Product: secreted levansucrase that renders
bacterial growth sensitive to sucrose
negative selection marker
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
5171 .. 6253  =  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
5171 .. 6253  =  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
7576 .. 8370  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
7576 .. 8370  =  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
4076 .. 4735  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
4076 .. 4735  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
6810 .. 7398  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6810 .. 7398  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
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
4992 .. 5078  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
4992 .. 5078  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
lacI promoter
6254 .. 6331  =  78 bp
lacI promoter
6254 .. 6331  =  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
3992 .. 4022  =  31 bp
   Segment 1:  -35  
   3992 .. 3997  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
3992 .. 4022  =  31 bp
   Segment 2:  
   3998 .. 4015  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
3992 .. 4022  =  31 bp
   Segment 3:  -10  
   4016 .. 4022  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
3992 .. 4022  =  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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