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Plasmid Files

pVP33K

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

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pVP33K Sequence and MappVP33K.dna
Map and Sequence File   
Sequence Author:  Center for Eukaryotic Structural Genomics
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 EagI (7040) NaeI (6448) NgoMIV (6446) RsrII (6431) AvrII (6279) PciI (5459) NdeI (5282) BstZ17I (5232) FspAI (5039) PpuMI * (5022) BstEII (4387) EcoRV (4123) HpaI (4067) PaeR7I - XhoI (117) AanI (165) MfeI (175) lac operator RBS BseRI (231) ATG NsiI (263) BsaI (327) AanI (616) BmgBI (806) AfeI (1174) PacI (1369) tetracysteine tag AsiSI - SgfI (1438) PmeI (2564) Eco53kI (2577) SacI (2579) SbfI (2612) BsrGI (2721) AscI (2738) SgrDI (2753) BspDI - ClaI (2766) BstBI (2777) HindIII (2780) NheI (2900) BmtI (2904) pVP33K 7253 bp
EagI  (7040)
1 site
C G G C C G G C C G G C
NaeI  (6448)
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  (6446)
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  (6431)
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  (6279)
1 site
C C T A G G G G A T C C
PciI  (5459)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NdeI  (5282)
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  (5232)
1 site
G T A T A C C A T A T G
FspAI  (5039)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (5022)
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  (4387)
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  (4123)
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  (4067)
1 site
G T T A A C C A A T T G
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  (263)
1 site
A T G C A T T A C G T A
BsaI  (327)
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  (616)
2 sites
T T A T A A A A T A T T
BmgBI  (806)
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  (1174)
1 site
A G C G C T T C G C G A
PacI  (1369)
1 site
T T A A T T A A A A T T A A T T
AsiSI  (1438)
1 site
G C G A T C G C C G C T A G C G
SgfI  (1438)
1 site
G C G A T C G C C G C T A G C G
PmeI  (2564)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (2577)
1 site
G A G C T C C T C G A G
SacI  (2579)
1 site
G A G C T C C T C G A G
SbfI  (2612)
1 site
C C T G C A G G G G A C G T C C
BsrGI  (2721)
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  (2738)
1 site
G G C G C G C C C C G C G C G G
SgrDI  (2753)
1 site
C G T C G A C G G C A G C T G C
BspDI  (2766)
1 site
A T C G A T T A G C T A
ClaI  (2766)
1 site
A T C G A T T A G C T A
BstBI  (2777)
1 site
T T C G A A A A G C T T
HindIII  (2780)
1 site
A A G C T T T T C G A A
NheI  (2900)
1 site
G C T A G C C G A T C G
BmtI  (2904)
1 site
G C T A G C C G A T C G
MBP
267 .. 1364  =  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
267 .. 1364  =  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
3842 .. 4924  =  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
3842 .. 4924  =  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
6285 .. 7079  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
6285 .. 7079  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
CmR
1900 .. 2559  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1900 .. 2559  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
2939 .. 3598  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
2939 .. 3598  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
5520 .. 6108  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5520 .. 6108  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
barnase
1465 .. 1800  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
barnase
1465 .. 1800  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
lambda t0 terminator
2801 .. 2895  =  95 bp
transcription terminator from phage lambda
lambda t0 terminator
2801 .. 2895  =  95 bp
transcription terminator from phage lambda
rrnB T1 terminator
3663 .. 3749  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
3663 .. 3749  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
lacIq promoter
4925 .. 5002  =  78 bp
In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about
10-fold.
lacIq promoter
4925 .. 5002  =  78 bp
In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about
10-fold.
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
1816 .. 1846  =  31 bp
   Segment 1:  -35  
   1816 .. 1821  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1816 .. 1846  =  31 bp
   Segment 2:  
   1822 .. 1839  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1816 .. 1846  =  31 bp
   Segment 3:  -10  
   1840 .. 1846  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1816 .. 1846  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
8xHis
237 .. 260  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
237 .. 260  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
HRV 3C site
1374 .. 1397  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human
rhinovirus 3C and PreScission proteases
HRV 3C site
1374 .. 1397  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human
rhinovirus 3C and PreScission proteases
TEV site
1416 .. 1436  =  21 bp
7 amino acids  =  884.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
1416 .. 1436  =  21 bp
7 amino acids  =  884.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
tetracysteine tag
1398 .. 1415  =  18 bp
6 amino acids  =  584.8 Da
Product: tetracysteine peptide that binds biarsenical
labeling reagents
tetracysteine tag
1398 .. 1415  =  18 bp
6 amino acids  =  584.8 Da
Product: tetracysteine peptide that binds biarsenical
labeling reagents
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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