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pMCSG31

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

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pMCSG31 Sequence and MappMCSG31.dna
Map and Sequence File   
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 (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) 6xHis 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

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.
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

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
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.
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

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
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.
AmpR
3520 .. 4380  =  861 bp
286 amino acids  =  31.5 kDa
   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
   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
TVMV site
2469 .. 2489  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa
protease recognition and cleavage site
TVMV site
2469 .. 2489  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa
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
TEV site
2532 .. 2552  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
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
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
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
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