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.

Sequence Author: Center for Eukaryotic Structural Genomics

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PaeR7I - XhoI (117) 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) AanI (165) MfeI (175) lac operator RBS BseRI (231) ATG NsiI (263) BsaI (327) AanI (616) BmgBI (806) AfeI (1174) PacI (1369) tetracysteine tag TEV site AsiSI - SgfI (1438) barnase 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
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
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
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
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
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
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.
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
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
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
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
4 segments
   Segment 1:  
   126 .. 140  =  15 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
126 .. 170  =  45 bp
4 segments
   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
4 segments
   Segment 3:  
   147 .. 163  =  17 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
126 .. 170  =  45 bp
4 segments
   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
3 segments
   Segment 1:  -35  
   1816 .. 1821  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1816 .. 1846  =  31 bp
3 segments
   Segment 2:  
   1822 .. 1839  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1816 .. 1846  =  31 bp
3 segments
   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
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
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).
ORF:  1465 .. 1800  =  336 bp
ORF:  111 amino acids  =  12.5 kDa
ORF:  1900 .. 2559  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  3808 .. 4059  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  2939 .. 3598  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  231 .. 1451  =  1221 bp
ORF:  406 amino acids  =  44.8 kDa
ORF:  6516 .. 6770  =  255 bp
ORF:  84 amino acids  =  9.8 kDa
ORF:  3822 .. 4085  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4674 .. 4991  =  318 bp
ORF:  105 amino acids  =  11.2 kDa
ORF:  6285 .. 7079  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  3842 .. 4801  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  6521 .. 6907  =  387 bp
ORF:  128 amino acids  =  14.7 kDa
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