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Plasmid Files

pVP68K

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

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pVP68K Sequence and MappVP68K.dna
Map and Sequence File   
Sequence Author:  Center for Eukaryotic Structural Genomics
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 FspI (5948) NaeI (5548) NgoMIV (5546) RsrII (5531) AvrII (5379) PciI (4558) NdeI (4381) BstZ17I (4331) BstEII (3525) EcoRV (3261) PaeR7I - XhoI (117) AanI (165) MfeI (175) lac operator RBS NcoI (229) BseRI (231) ATG NsiI (263) BsaI (327) AanI (616) BmgBI (806) AfeI (1174) PacI (1369) AsiSI - SgfI (1417) NotI (1420) Bpu10I (1871) PasI (2330) SspI (2410) ScaI (2515) PmeI (2549) Eco53kI (2562) SacI (2564) SbfI (2597) BsrGI (2703) AscI (2720) SgrDI (2735) BspDI - ClaI (2748) BstBI (2759) HindIII (2762) HpaI (3205) pVP68K 6353 bp
FspI  (5948)
1 site
T G C G C A A C G C G T
NaeI  (5548)
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  (5546)
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  (5531)
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  (5379)
1 site
C C T A G G G G A T C C
PciI  (4558)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NdeI  (4381)
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  (4331)
1 site
G T A T A C C A T A T G
BstEII  (3525)
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  (3261)
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.
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
NcoI  (229)
1 site
C C A T G G G G T A C 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  (1417)
1 site
G C G A T C G C C G C T A G C G
SgfI  (1417)
1 site
G C G A T C G C C G C T A G C G
NotI  (1420)
1 site
G C G G C C G C C G C C G G C G
Bpu10I  (1871)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends
generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
PasI  (2330)
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.
SspI  (2410)
1 site
A A T A T T T T A T A A
ScaI  (2515)
1 site
A G T A C T T C A T G A
PmeI  (2549)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (2562)
1 site
G A G C T C C T C G A G
SacI  (2564)
1 site
G A G C T C C T C G A G
SbfI  (2597)
1 site
C C T G C A G G G G A C G T C C
BsrGI  (2703)
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  (2720)
1 site
G G C G C G C C C C G C G C G G
SgrDI  (2735)
1 site
C G T C G A C G G C A G C T G C
BspDI  (2748)
1 site
A T C G A T T A G C T A
ClaI  (2748)
1 site
A T C G A T T A G C T A
BstBI  (2759)
1 site
T T C G A A A A G C T T
HindIII  (2762)
1 site
A A G C T T T T C G A A
HpaI  (3205)
1 site
G T T A A C C A A T T 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
2980 .. 4062  =  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
2980 .. 4062  =  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
5385 .. 6179  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
5385 .. 6179  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
CmR
1885 .. 2544  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1885 .. 2544  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
4619 .. 5207  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4619 .. 5207  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
barnase
1450 .. 1785  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
barnase
1450 .. 1785  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
rrnB T1 terminator
2801 .. 2887  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
2801 .. 2887  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
lacI promoter
4063 .. 4140  =  78 bp
lacI promoter
4063 .. 4140  =  78 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
1801 .. 1831  =  31 bp
   Segment 1:  -35  
   1801 .. 1806  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1801 .. 1831  =  31 bp
   Segment 2:  
   1807 .. 1824  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1801 .. 1831  =  31 bp
   Segment 3:  -10  
   1825 .. 1831  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1801 .. 1831  =  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
1389 .. 1412  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human
rhinovirus 3C and PreScission proteases
HRV 3C site
1389 .. 1412  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human
rhinovirus 3C and PreScission proteases
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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