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Plasmid Files

pVP16

Bacterial Gateway® destination vector for appending an N-terminal 8xHis-MBP cassette to an expressed protein.

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pVP16 Sequence and MappVP16.dna
Map and Sequence File   
Sequence Author:  Center for Eukaryotic Structural Genomics
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 PaeR7I - XhoI (2) XmnI (7238) BglI (6759) AhdI (6639) PciI (5746) BspQI - SapI (5630) NdeI (5569) PflFI - Tth111I (5493) FspAI (5326) PpuMI * (5309) BstEII (4674) ApaI (4653) PspOMI (4649) EcoRV (4410) HpaI (4354) PluTI (4221) SfoI (4219) NarI * (4218) KasI (4217) T5 promoter MfeI (60) lac operator RBS BseRI (116) ATG BsiWI (436) AfeI (1056) Eco53kI (1254) SacI (1256) EagI - NotI (1442) BbvCI (2487) TspMI - XmaI (2631) SmaI - SrfI (2633) PstI (2888) AscI (3025) SgrDI (3040) Acc65I (3046) KpnI (3050) BspDI - ClaI (3053) BstBI (3064) HindIII (3067) NheI (3187) BmtI (3191) pVP16 7631 bp
PaeR7I  (2)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (2)
1 site
C T C G A G G A G C T C
XmnI  (7238)
1 site
G A A N N N N T T C C T T N N N N A A G
BglI  (6759)
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  (6639)
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  (5746)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (5630)
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  (5630)
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  (5569)
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.
PflFI  (5493)
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  (5493)
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  (5326)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (5309)
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  (4674)
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  (4653)
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  (4649)
1 site
G G G C C C C C C G G G
EcoRV  (4410)
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  (4354)
1 site
G T T A A C C A A T T G
PluTI  (4221)
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  (4219)
1 site
G G C G C C C C G C G G
NarI  (4218)
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.
KasI  (4217)
1 site
G G C G C C C C G C G G
MfeI  (60)
1 site
C A A T T G G T T A A C
BseRI  (116)
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.
BsiWI  (436)
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.
AfeI  (1056)
1 site
A G C G C T T C G C G A
Eco53kI  (1254)
1 site
G A G C T C C T C G A G
SacI  (1256)
1 site
G A G C T C C T C G A G
EagI  (1442)
1 site
C G G C C G G C C G G C
NotI  (1442)
1 site
G C G G C C G C C G C C G G C G
BbvCI  (2487)
1 site
C C T C A G C G G A G T C G
TspMI  (2631)
1 site
C C C G G G G G G C C C
XmaI  (2631)
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.
SmaI  (2633)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (2633)
1 site
G C C C G G G C C G G G C C C G
PstI  (2888)
1 site
C T G C A G G A C G T C
AscI  (3025)
1 site
G G C G C G C C C C G C G C G G
SgrDI  (3040)
1 site
C G T C G A C G G C A G C T G C
Acc65I  (3046)
1 site
G G T A C C C C A T G G
KpnI  (3050)
1 site
G G T A C C C C A T G G
BspDI  (3053)
1 site
A T C G A T T A G C T A
ClaI  (3053)
1 site
A T C G A T T A G C T A
BstBI  (3064)
1 site
T T C G A A A A G C T T
HindIII  (3067)
1 site
A A G C T T T T C G A A
NheI  (3187)
1 site
G C T A G C C G A T C G
BmtI  (3191)
1 site
G C T A G C C G A T C G
MBP
149 .. 1246  =  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
149 .. 1246  =  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
4129 .. 5211  =  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
4129 .. 5211  =  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
6566 .. 7426  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6566 .. 7357  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6566 .. 7426  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7358 .. 7426  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6566 .. 7426  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
1549 .. 2208  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1549 .. 2208  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
3226 .. 3885  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
3226 .. 3885  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
5807 .. 6395  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5807 .. 6395  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ccdB
2550 .. 2855  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
2550 .. 2855  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
attR1
1316 .. 1440  =  125 bp
recombination site for the Gateway® LR reaction
attR1
1316 .. 1440  =  125 bp
recombination site for the Gateway® LR reaction
attR2
2896 .. 3020  =  125 bp
recombination site for the Gateway® LR reaction
attR2
2896 .. 3020  =  125 bp
recombination site for the Gateway® LR reaction
AmpR promoter
7427 .. 7531  =  105 bp
AmpR promoter
7427 .. 7531  =  105 bp
lambda t0 terminator
3088 .. 3182  =  95 bp
transcription terminator from phage lambda
lambda t0 terminator
3088 .. 3182  =  95 bp
transcription terminator from phage lambda
rrnB T1 terminator
3950 .. 4036  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
3950 .. 4036  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
lacIq promoter
5212 .. 5289  =  78 bp
In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about
10-fold.
lacIq promoter
5212 .. 5289  =  78 bp
In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about
10-fold.
T5 promoter
11 .. 55  =  45 bp
   Segment 1:  
   11 .. 25  =  15 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
11 .. 55  =  45 bp
   Segment 2:  -35  
   26 .. 31  =  6 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
11 .. 55  =  45 bp
   Segment 3:  
   32 .. 48  =  17 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
11 .. 55  =  45 bp
   Segment 4:  -10  
   49 .. 55  =  7 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
11 .. 55  =  45 bp
4 segments
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
lac UV5 promoter
1465 .. 1495  =  31 bp
   Segment 1:  -35  
   1465 .. 1470  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1465 .. 1495  =  31 bp
   Segment 2:  
   1471 .. 1488  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1465 .. 1495  =  31 bp
   Segment 3:  -10  
   1489 .. 1495  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1465 .. 1495  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
8xHis
122 .. 145  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
122 .. 145  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
lac operator
63 .. 79  =  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
63 .. 79  =  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
102 .. 107  =  6 bp
ribosome binding site
RBS
102 .. 107  =  6 bp
ribosome binding site
ATG
116 .. 118  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
116 .. 118  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
lac operator
31 .. 47  =  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
31 .. 47  =  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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