pVP56A

Bacterial Flexi® Vector with an ampicillin resistance marker, for appending an N-terminal 8xHis-MBP-HRV 3C-TEV cassette to an expressed protein.

Sequence Author: Center for Eukaryotic Structural Genomics

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PaeR7I - XhoI (37) XmnI (6865) BglI (6386) AhdI (6266) PciI (5373) BspQI - SapI (5257) NdeI (5196) BstZ17I (5146) PflFI - Tth111I (5120) FspAI (4953) PpuMI * (4936) BstEII (4301) EcoRV (4037) HpaI (3981) PluTI (3848) SfoI (3846) NarI * (3845) T5 promoter AanI (85) MfeI (95) lac operator RBS BseRI (151) ATG NsiI (183) BsiWI (474) AanI (536) BglII (543) BmgBI (726) BsaBI (859) AfeI (1094) PacI (1289) TEV site AsiSI - SgfI (1355) PmeI (2481) Eco53kI (2494) SacI (2496) PstI - SbfI (2529) SphI (2535) BsrGI (2635) AscI (2652) SgrDI (2667) BspDI - ClaI (2680) BstBI (2691) HindIII (2694) NheI (2812) BmtI (2816) KasI (3844) pVP56A 7222 bp
PaeR7I  (37)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (37)
1 site
C T C G A G G A G C T C
XmnI  (6865)
1 site
G A A N N N N T T C C T T N N N N A A G
BglI  (6386)
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.
AhdI  (6266)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
PciI  (5373)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (5257)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (5257)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be mixed before removing an aliquot.
NdeI  (5196)
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  (5146)
1 site
G T A T A C C A T A T G
PflFI  (5120)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (5120)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
FspAI  (4953)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (4936)
1 site
R G G W C C Y Y C C W G G R
* Blocked by Dcm methylation.
Sticky ends from different PpuMI sites may not be compatible.
BstEII  (4301)
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.
EcoRV  (4037)
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  (3981)
1 site
G T T A A C C A A T T G
PluTI  (3848)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
SfoI  (3846)
1 site
G G C G C C C C G C G G
NarI  (3845)
1 site
G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI recognition sequence.
AanI  (85)
2 sites
T T A T A A A A T A T T
MfeI  (95)
1 site
C A A T T G G T T A A C
BseRI  (151)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
NsiI  (183)
1 site
A T G C A T T A C G T A
BsiWI  (474)
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.
AanI  (536)
2 sites
T T A T A A A A T A T T
BglII  (543)
1 site
A G A T C T T C T A G A
BmgBI  (726)
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.
BsaBI  (859)
1 site
G A T N N N N A T C C T A N N N N T A G
AfeI  (1094)
1 site
A G C G C T T C G C G A
PacI  (1289)
1 site
T T A A T T A A A A T T A A T T
AsiSI  (1355)
1 site
G C G A T C G C C G C T A G C G
SgfI  (1355)
1 site
G C G A T C G C C G C T A G C G
PmeI  (2481)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (2494)
1 site
G A G C T C C T C G A G
SacI  (2496)
1 site
G A G C T C C T C G A G
PstI  (2529)
1 site
C T G C A G G A C G T C
SbfI  (2529)
1 site
C C T G C A G G G G A C G T C C
SphI  (2535)
1 site
G C A T G C C G T A C G
BsrGI  (2635)
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  (2652)
1 site
G G C G C G C C C C G C G C G G
SgrDI  (2667)
1 site
C G T C G A C G G C A G C T G C
BspDI  (2680)
1 site
A T C G A T T A G C T A
ClaI  (2680)
1 site
A T C G A T T A G C T A
BstBI  (2691)
1 site
T T C G A A A A G C T T
HindIII  (2694)
1 site
A A G C T T T T C G A A
NheI  (2812)
1 site
G C T A G C C G A T C G
BmtI  (2816)
1 site
G C T A G C C G A T C G
KasI  (3844)
1 site
G G C G C C C C G C G G
ATG
151 .. 153  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
151 .. 153  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
8xHis
157 .. 180  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
157 .. 180  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
MBP
187 .. 1284  =  1098 bp
366 amino acids  =  40.2 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
187 .. 1284  =  1098 bp
366 amino acids  =  40.2 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.
HRV 3C site
1309 .. 1332  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
HRV 3C site
1309 .. 1332  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
TEV site
1333 .. 1353  =  21 bp
7 amino acids  =  884.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
1333 .. 1353  =  21 bp
7 amino acids  =  884.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
lacI
3756 .. 4838  =  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
3756 .. 4838  =  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).
AmpR
6193 .. 7053  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   6193 .. 6984  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6193 .. 7053  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   6985 .. 7053  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6193 .. 7053  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
CmR
1817 .. 2476  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1817 .. 2476  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
5434 .. 6022  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5434 .. 6022  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
barnase
1382 .. 1717  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus amyloliquefaciens
The barnase gene is lethal in standard bacterial transformation strains.
barnase
1382 .. 1717  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus amyloliquefaciens
The barnase gene is lethal in standard bacterial transformation strains.
AmpR promoter
7054 .. 7158  =  105 bp
AmpR promoter
7054 .. 7158  =  105 bp
lambda t0 terminator
2713 .. 2807  =  95 bp
transcription terminator from phage lambda
lambda t0 terminator
2713 .. 2807  =  95 bp
transcription terminator from phage lambda
rrnB T1 terminator
3577 .. 3663  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
3577 .. 3663  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
lacIq promoter
4839 .. 4916  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
lacIq promoter
4839 .. 4916  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
T5 promoter
46 .. 90  =  45 bp
4 segments
   Segment 1:  
   46 .. 60  =  15 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
46 .. 90  =  45 bp
4 segments
   Segment 2:  -35  
   61 .. 66  =  6 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
46 .. 90  =  45 bp
4 segments
   Segment 3:  
   67 .. 83  =  17 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
46 .. 90  =  45 bp
4 segments
   Segment 4:  -10  
   84 .. 90  =  7 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
46 .. 90  =  45 bp
4 segments
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
lac UV5 promoter
1733 .. 1763  =  31 bp
3 segments
   Segment 1:  -35  
   1733 .. 1738  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1733 .. 1763  =  31 bp
3 segments
   Segment 2:  
   1739 .. 1756  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1733 .. 1763  =  31 bp
3 segments
   Segment 3:  -10  
   1757 .. 1763  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1733 .. 1763  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
lac operator
98 .. 114  =  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
98 .. 114  =  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
137 .. 142  =  6 bp
ribosome binding site
RBS
137 .. 142  =  6 bp
ribosome binding site
lac operator
66 .. 82  =  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
66 .. 82  =  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).
ORF:  151 .. 1368  =  1218 bp
ORF:  405 amino acids  =  44.5 kDa
ORF:  2851 .. 3093  =  243 bp
ORF:  80 amino acids  =  9.5 kDa
ORF:  1382 .. 1717  =  336 bp
ORF:  111 amino acids  =  12.5 kDa
ORF:  1817 .. 2476  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  3722 .. 3973  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  6323 .. 6589  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3276 .. 3512  =  237 bp
ORF:  78 amino acids  =  8.9 kDa
ORF:  3736 .. 3999  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4588 .. 4905  =  318 bp
ORF:  105 amino acids  =  11.2 kDa
ORF:  6193 .. 7053  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  3756 .. 4715  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
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