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Plasmid Files

pVP33A

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

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pVP33A Sequence and MappVP33A.dna
Map and Sequence File   
Sequence Author:  Center for Eukaryotic Structural Genomics
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 PaeR7I - XhoI (37) XmnI (6871) BglI (6392) AhdI (6272) PciI (5379) BspQI - SapI (5263) NdeI (5202) BstZ17I (5152) PflFI - Tth111I (5126) FspAI (4959) PpuMI * (4942) BstEII (4307) EcoRV (4043) HpaI (3987) PluTI (3854) SfoI (3852) NarI * (3851) 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) tetracysteine tag AsiSI - SgfI (1358) PmeI (2484) Eco53kI (2497) SacI (2499) PstI - SbfI (2532) SphI (2538) BsrGI (2641) AscI (2658) SgrDI (2673) BspDI - ClaI (2686) BstBI (2697) HindIII (2700) NheI (2818) BmtI (2822) KasI (3850) pVP33A 7228 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  (6871)
1 site
G A A N N N N T T C C T T N N N N A A G
BglI  (6392)
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  (6272)
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  (5379)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (5263)
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  (5263)
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  (5202)
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  (5152)
1 site
G T A T A C C A T A T G
PflFI  (5126)
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  (5126)
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  (4959)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (4942)
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  (4307)
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  (4043)
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  (3987)
1 site
G T T A A C C A A T T G
PluTI  (3854)
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  (3852)
1 site
G G C G C C C C G C G G
NarI  (3851)
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  (1358)
1 site
G C G A T C G C C G C T A G C G
SgfI  (1358)
1 site
G C G A T C G C C G C T A G C G
PmeI  (2484)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (2497)
1 site
G A G C T C C T C G A G
SacI  (2499)
1 site
G A G C T C C T C G A G
PstI  (2532)
1 site
C T G C A G G A C G T C
SbfI  (2532)
1 site
C C T G C A G G G G A C G T C C
SphI  (2538)
1 site
G C A T G C C G T A C G
BsrGI  (2641)
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  (2658)
1 site
G G C G C G C C C C G C G C G G
SgrDI  (2673)
1 site
C G T C G A C G G C A G C T G C
BspDI  (2686)
1 site
A T C G A T T A G C T A
ClaI  (2686)
1 site
A T C G A T T A G C T A
BstBI  (2697)
1 site
T T C G A A A A G C T T
HindIII  (2700)
1 site
A A G C T T T T C G A A
NheI  (2818)
1 site
G C T A G C C G A T C G
BmtI  (2822)
1 site
G C T A G C C G A T C G
KasI  (3850)
1 site
G G C G C C C C G C G G
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.
lacI
3762 .. 4844  =  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
3762 .. 4844  =  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
6199 .. 7059  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6199 .. 6990  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6199 .. 7059  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6991 .. 7059  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6199 .. 7059  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
1820 .. 2479  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1820 .. 2479  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
5440 .. 6028  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5440 .. 6028  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
barnase
1385 .. 1720  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
barnase
1385 .. 1720  =  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
7060 .. 7164  =  105 bp
AmpR promoter
7060 .. 7164  =  105 bp
lambda t0 terminator
2719 .. 2813  =  95 bp
transcription terminator from phage lambda
lambda t0 terminator
2719 .. 2813  =  95 bp
transcription terminator from phage lambda
rrnB T1 terminator
3583 .. 3669  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
3583 .. 3669  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
lacIq promoter
4845 .. 4922  =  78 bp
In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about
10-fold.
lacIq promoter
4845 .. 4922  =  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
   Segment 1:  
   46 .. 60  =  15 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
46 .. 90  =  45 bp
   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
   Segment 3:  
   67 .. 83  =  17 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
46 .. 90  =  45 bp
   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
1736 .. 1766  =  31 bp
   Segment 1:  -35  
   1736 .. 1741  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1736 .. 1766  =  31 bp
   Segment 2:  
   1742 .. 1759  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1736 .. 1766  =  31 bp
   Segment 3:  -10  
   1760 .. 1766  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1736 .. 1766  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
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
HRV 3C site
1294 .. 1317  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human
rhinovirus 3C and PreScission proteases
HRV 3C site
1294 .. 1317  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human
rhinovirus 3C and PreScission proteases
TEV site
1336 .. 1356  =  21 bp
7 amino acids  =  884.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
1336 .. 1356  =  21 bp
7 amino acids  =  884.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
tetracysteine tag
1318 .. 1335  =  18 bp
6 amino acids  =  584.8 Da
Product: tetracysteine peptide that binds biarsenical
labeling reagents
tetracysteine tag
1318 .. 1335  =  18 bp
6 amino acids  =  584.8 Da
Product: tetracysteine peptide that binds biarsenical
labeling reagents
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
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
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).
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