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Plasmid Files

pVP91A

Bacterial Flexi® Vector with an ampicillin resistance marker, for appending an N-terminal 8xHis tag to an expressed protein.

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pVP91A Sequence and MappVP91A.dna
Map and Sequence File   
Sequence Author:  Center for Eukaryotic Structural Genomics
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 EcoO109I (100) BsiWI (6529) FspI (5887) BglI (5785) BsaI (5726) AhdI (5665) AvrII (5587) AlwNI (5183) BspQI - SapI (4651) NdeI (4590) PflFI - Tth111I (4514) PfoI (4411) BclI * (3902) BstEII (3734) ApaI (3713) PspOMI (3709) PluTI (3281) SfoI (3279) NarI * (3278) PaeR7I - XhoI (117) AanI (165) MfeI (175) lac operator RBS BseRI (231) ATG NsiI (235) 8xHis AsiSI - SgfI (262) StuI * (744) AanI (1143) SnaBI (1252) BtgI (1579) SacII (1582) BspEI (2303) PasI (2539) PmeI (2758) Eco53kI (2771) SacI (2773) TspMI - XmaI (2779) SmaI (2781) PstI - SbfI (2806) BfuAI - BspMI (2809) SphI (2812) BlpI (2871) AscI (2929) SgrDI (2944) BstBI (2968) KasI (3277) pVP91A 6534 bp
EcoO109I  (100)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
BsiWI  (6529)
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.
FspI  (5887)
1 site
T G C G C A A C G C G T
BglI  (5785)
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.
BsaI  (5726)
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.
AhdI  (5665)
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.
AvrII  (5587)
1 site
C C T A G G G G A T C C
AlwNI  (5183)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
BspQI  (4651)
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  (4651)
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  (4590)
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  (4514)
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  (4514)
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.
PfoI  (4411)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
BclI  (3902)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
BstEII  (3734)
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  (3713)
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  (3709)
1 site
G G G C C C C C C G G G
PluTI  (3281)
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  (3279)
1 site
G G C G C C C C G C G G
NarI  (3278)
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.
PaeR7I  (117)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (117)
1 site
C T C G A G G A G C T C
AanI  (165)
2 sites
T T A T A A A A T A T T
MfeI  (175)
1 site
C A A T T G G T T A A C
BseRI  (231)
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  (235)
1 site
A T G C A T T A C G T A
AsiSI  (262)
1 site
G C G A T C G C C G C T A G C G
SgfI  (262)
1 site
G C G A T C G C C G C T A G C G
StuI  (744)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
AanI  (1143)
2 sites
T T A T A A A A T A T T
SnaBI  (1252)
1 site
T A C G T A A T G C A T
BtgI  (1579)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
SacII  (1582)
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.
BspEI  (2303)
1 site
T C C G G A A G G C C T
PasI  (2539)
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.
PmeI  (2758)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (2771)
1 site
G A G C T C C T C G A G
SacI  (2773)
1 site
G A G C T C C T C G A G
TspMI  (2779)
1 site
C C C G G G G G G C C C
XmaI  (2779)
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  (2781)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
PstI  (2806)
1 site
C T G C A G G A C G T C
SbfI  (2806)
1 site
C C T G C A G G G G A C G T C C
BfuAI  (2809)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (2809)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
SphI  (2812)
1 site
G C A T G C C G T A C G
BlpI  (2871)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
AscI  (2929)
1 site
G G C G C G C C C C G C G C G G
SgrDI  (2944)
1 site
C G T C G A C G G C A G C T G C
BstBI  (2968)
1 site
T T C G A A A A G C T T
KasI  (3277)
1 site
G G C G C C C C G C G G
SacB
503 .. 1924  =  1422 bp
473 amino acids  =  53.0 kDa
   Segment 1:  signal peptide  
   503 .. 589  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders
bacterial growth sensitive to sucrose
negative selection marker
SacB
503 .. 1924  =  1422 bp
473 amino acids  =  53.0 kDa
   Segment 2:  
   590 .. 1924  =  1335 bp
   444 amino acids  =  50.0 kDa
Product: secreted levansucrase that renders
bacterial growth sensitive to sucrose
negative selection marker
SacB
503 .. 1924  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
Product: secreted levansucrase that renders
bacterial growth sensitive to sucrose
negative selection marker
lacI
3189 .. 4271  =  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
3189 .. 4271  =  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
5595 .. 6452  =  858 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5595 .. 6383  =  789 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5595 .. 6452  =  858 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6384 .. 6452  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5595 .. 6452  =  858 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
2094 .. 2753  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
2094 .. 2753  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
4828 .. 5416  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4828 .. 5416  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rrnB T1 terminator
3010 .. 3096  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
3010 .. 3096  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
lacI promoter
4272 .. 4349  =  78 bp
lacI promoter
4272 .. 4349  =  78 bp
AmpR promoter
6453 .. 6524  =  72 bp
AmpR promoter
6453 .. 6524  =  72 bp
T5 promoter
126 .. 170  =  45 bp
   Segment 1:  
   126 .. 140  =  15 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
126 .. 170  =  45 bp
   Segment 2:  -35  
   141 .. 146  =  6 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
126 .. 170  =  45 bp
   Segment 3:  
   147 .. 163  =  17 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
126 .. 170  =  45 bp
   Segment 4:  -10  
   164 .. 170  =  7 bp
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
T5 promoter
126 .. 170  =  45 bp
4 segments
bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator
lac UV5 promoter
2010 .. 2040  =  31 bp
   Segment 1:  -35  
   2010 .. 2015  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2010 .. 2040  =  31 bp
   Segment 2:  
   2016 .. 2033  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2010 .. 2040  =  31 bp
   Segment 3:  -10  
   2034 .. 2040  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2010 .. 2040  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
8xHis
234 .. 257  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
234 .. 257  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
lac operator
178 .. 194  =  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
178 .. 194  =  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
217 .. 222  =  6 bp
ribosome binding site
RBS
217 .. 222  =  6 bp
ribosome binding site
ATG
231 .. 233  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
231 .. 233  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
lac operator
146 .. 162  =  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
146 .. 162  =  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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