pMCSG31

Bacterial expression vector with an MBP-TVMV-6xHis-TEV leader and a Tet-inducible TVMV protease cassette.

Sequence Author: Midwest Center for Structural Genomics

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BsgI (4952) AleI (4928) NruI (4670) SnaBI (4627) EcoNI (4598) Bpu10I (4551) AhdI (4307) BpmI (4238) NmeAIII (4160) MfeI (4129) FspI (4084) ScaI (3826) XmnI (3707) BstBI (2834) PflMI (7) BseRI (246) AgeI (411) BsmBI - Esp3I (552) BbsI (596) AflIII - MluI (910) SphI (1031) SgrAI (1179) BspDI * - ClaI * (1223) T7 promoter RBS PfoI * (1588) BsiWI (1622) BglII (1691) BstXI (1717) BssHII (1768) BmgBI (1874) BsrGI (2449) 6xHis Acc65I (2526) KpnI (2530) XcmI (2553) SspI (2555) Eco53kI (2586) SacI (2588) EagI - NotI (2604) TspMI - XmaI (2768) SmaI (2770) AbsI (2779) SbfI (2795) Strep-Tag II pMCSG31 5822 bp
BsgI  (4952)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
AleI  (4928)
1 site
C A C N N N N G T G G T G N N N N C A C
NruI  (4670)
1 site
T C G C G A A G C G C T
SnaBI  (4627)
1 site
T A C G T A A T G C A T
EcoNI  (4598)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
Bpu10I  (4551)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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.
AhdI  (4307)
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.
BpmI  (4238)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
NmeAIII  (4160)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
MfeI  (4129)
1 site
C A A T T G G T T A A C
FspI  (4084)
1 site
T G C G C A A C G C G T
ScaI  (3826)
1 site
A G T A C T T C A T G A
XmnI  (3707)
1 site
G A A N N N N T T C C T T N N N N A A G
BstBI  (2834)
1 site
T T C G A A A A G C T T
PflMI  (7)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
BseRI  (246)
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.
AgeI  (411)
1 site
A C C G G T T G G C C A
BsmBI  (552)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (552)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I sites may not be compatible.
BbsI  (596)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
AflIII  (910)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
MluI  (910)
1 site
A C G C G T T G C G C A
SphI  (1031)
1 site
G C A T G C C G T A C G
SgrAI  (1179)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
BspDI  (1223)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1223)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
PfoI  (1588)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
BsiWI  (1622)
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.
BglII  (1691)
1 site
A G A T C T T C T A G A
BstXI  (1717)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
BssHII  (1768)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
BmgBI  (1874)
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.
BsrGI  (2449)
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.
Acc65I  (2526)
1 site
G G T A C C C C A T G G
KpnI  (2530)
1 site
G G T A C C C C A T G G
XcmI  (2553)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
SspI  (2555)
1 site
A A T A T T T T A T A A
Eco53kI  (2586)
1 site
G A G C T C C T C G A G
SacI  (2588)
1 site
G A G C T C C T C G A G
EagI  (2604)
1 site
C G G C C G G C C G G C
NotI  (2604)
1 site
G C G G C C G C C G C C G G C G
TspMI  (2768)
1 site
C C C G G G G G G C C C
XmaI  (2768)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (2770)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
AbsI  (2779)
1 site
C C T C G A G G G G A G C T C C
SbfI  (2795)
1 site
C C T G C A G G G G A C G T C C
MBP
1332 .. 2432  =  1101 bp
367 amino acids  =  40.3 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
1332 .. 2432  =  1101 bp
367 amino acids  =  40.3 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.
TVMV site
2469 .. 2489  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TVMV site
2469 .. 2489  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
6xHis
2490 .. 2507  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2490 .. 2507  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
TEV site
2532 .. 2552  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
2532 .. 2552  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
AmpR
3520 .. 4380  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   3520 .. 3588  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3520 .. 4380  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3589 .. 4380  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3520 .. 4380  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
TVMV protease
184 .. 894  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease (Nallamsetty et al., 2004)
TVMV protease
184 .. 894  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease (Nallamsetty et al., 2004)
TetR
4387 .. 5013  =  627 bp
208 amino acids  =  23.5 kDa
Product: tetracycline repressor TetR
TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline.
TetR
4387 .. 5013  =  627 bp
208 amino acids  =  23.5 kDa
Product: tetracycline repressor TetR
TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline.
ori
5166 .. 5754  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5166 .. 5754  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
2932 .. 3390  =  459 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2932 .. 3390  =  459 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
AmpR promoter
3416 .. 3519  =  104 bp
AmpR promoter
3416 .. 3519  =  104 bp
rrnB T1 terminator
927 .. 1013  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
927 .. 1013  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
tetR/tetA promoters
36 .. 91  =  56 bp
overlapping promoters for bacterial tetR and tetA
tetR/tetA promoters
36 .. 91  =  56 bp
overlapping promoters for bacterial tetR and tetA
T7 terminator
2702 .. 2749  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
2702 .. 2749  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
1263 .. 1287  =  25 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
1263 .. 1287  =  25 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).
Strep-Tag II
2818 .. 2841  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin, an engineered form of streptavidin
Strep-Tag II
2818 .. 2841  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin, an engineered form of streptavidin
T7 promoter
1244 .. 1262  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1244 .. 1262  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
2618 .. 2635  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2618 .. 2635  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
1318 .. 1323  =  6 bp
ribosome binding site
RBS
1318 .. 1323  =  6 bp
ribosome binding site
tet operator
42 .. 60  =  19 bp
bacterial operator O1 for the tetR and tetA genes
tet operator
42 .. 60  =  19 bp
bacterial operator O1 for the tetR and tetA genes
tet operator
72 .. 90  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
72 .. 90  =  19 bp
bacterial operator O2 for the tetR and tetA genes
ORF:  160 .. 894  =  735 bp
ORF:  244 amino acids  =  27.5 kDa
ORF:  3520 .. 4380  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  4387 .. 5013  =  627 bp
ORF:  208 amino acids  =  23.5 kDa
ORF:  761 .. 1006  =  246 bp
ORF:  81 amino acids  =  9.2 kDa
ORF:  1029 .. 1280  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  1332 .. 2570  =  1239 bp
ORF:  412 amino acids  =  45.3 kDa
ORF:  891 .. 1451  =  561 bp
ORF:  186 amino acids  =  20.2 kDa
ORF:  1671 .. 2504  =  834 bp
ORF:  277 amino acids  =  32.3 kDa
ORF:  3984 .. 4250  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  4311 .. 4559  =  249 bp
ORF:  82 amino acids  =  8.9 kDa
ORF:  5808 .. 585  =  600 bp
ORF:  199 amino acids  =  23.2 kDa
ORF:  2440 .. 2889  =  450 bp
ORF:  149 amino acids  =  16.4 kDa
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