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Plasmid Files

pMCSG35

Bacterial vector encoding N-terminal 10xHis-MBP-TEV and C-terminal TEV-6xHis plus TVMV protease. See also pMCSG35B.

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pMCSG35 Sequence and MappMCSG35.dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 6xHis ScaI (6789) AhdI (6309) PciI (5416) BspQI - SapI (5300) BstZ17I (5187) PflFI - Tth111I (5161) Bpu10I (4522) PpuMI (4422) FspAI (4397) PshAI (4160) HpaI (3821) EagI - NotI (166) SalI (179) Eco53kI (188) SacI (190) BamHI (198) 6xHis TEV site TspMI - XmaI (253) SmaI (255) TEV site BsmI (786) BmgBI (862) PsiI (1052) BsiWI (1110) ATG RBS T7 promoter BspDI * - ClaI * (1565) SgrAI (1607) SphI (1763) ZraI (1767) AatII (1769) BseRI (2005) NsiI (2127) AgeI (2170) DraIII (2338) SpeI (2407) SacII (2526) SphI (2790) EcoNI (2850) PflMI (2897) BstEII (3496) PspOMI (3522) ApaI (3526) pMCSG35 7484 bp
ScaI  (6789)
1 site
A G T A C T T C A T G A
AhdI  (6309)
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.
PciI  (5416)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (5300)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (5300)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
BstZ17I  (5187)
1 site
G T A T A C C A T A T G
PflFI  (5161)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (5161)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
Bpu10I  (4522)
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.
PpuMI  (4422)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
FspAI  (4397)
1 site
R T G C G C A Y Y A C G C G T R
PshAI  (4160)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
HpaI  (3821)
1 site
G T T A A C C A A T T G
EagI  (166)
1 site
C G G C C G G C C G G C
NotI  (166)
1 site
G C G G C C G C C G C C G G C G
SalI  (179)
1 site
G T C G A C C A G C T G
Eco53kI  (188)
1 site
G A G C T C C T C G A G
SacI  (190)
1 site
G A G C T C C T C G A G
BamHI  (198)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can
remain bound to DNA and alter its electrophoretic mobility.
TspMI  (253)
1 site
C C C G G G G G G C C C
XmaI  (253)
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  (255)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BsmI  (786)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
BmgBI  (862)
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.
PsiI  (1052)
1 site
T T A T A A A A T A T T
BsiWI  (1110)
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.
BspDI  (1565)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1565)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (1607)
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.
SphI  (1763)
2 sites
G C A T G C C G T A C G
ZraI  (1767)
1 site
G A C G T C C T G C A G
AatII  (1769)
1 site
G A C G T C C T G C A G
BseRI  (2005)
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  (2127)
1 site
A T G C A T T A C G T A
AgeI  (2170)
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.
DraIII  (2338)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
SpeI  (2407)
1 site
A C T A G T T G A T C A
SacII  (2526)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
SphI  (2790)
2 sites
G C A T G C C G T A C G
EcoNI  (2850)
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.
PflMI  (2897)
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.
BstEII  (3496)
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.
PspOMI  (3522)
1 site
G G G C C C C C C G G G
ApaI  (3526)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
MBP
305 .. 1402  =  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
305 .. 1402  =  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
2965 .. 4047  =  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
2965 .. 4047  =  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).
AmpR
6236 .. 7096  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   6236 .. 7027  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6236 .. 7096  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   7028 .. 7096  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6236 .. 7096  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TVMV protease
1943 .. 2653  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease
(Nallamsetty et al., 2004)
TVMV protease
1943 .. 2653  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease
(Nallamsetty et al., 2004)
ori
5477 .. 6065  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5477 .. 6065  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
4856 .. 5047  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
4856 .. 5047  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
7097 .. 7200  =  104 bp
AmpR promoter
7097 .. 7200  =  104 bp
rrnB T1 terminator
2686 .. 2772  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
2686 .. 2772  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
lacI promoter
2887 .. 2964  =  78 bp
lacI promoter
2887 .. 2964  =  78 bp
PLtetO-1 promoter
1846 .. 1919  =  74 bp
   Segment 1:  
   1846 .. 1864  =  19 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1846 .. 1919  =  74 bp
   Segment 2:  -35  
   1865 .. 1870  =  6 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1846 .. 1919  =  74 bp
   Segment 3:  
   1871 .. 1887  =  17 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1846 .. 1919  =  74 bp
   Segment 4:  -10  
   1888 .. 1893  =  6 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1846 .. 1919  =  74 bp
   Segment 5:  
   1894 .. 1919  =  26 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1846 .. 1919  =  74 bp
5 segments
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
10xHis
1427 .. 1456  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
10xHis
1427 .. 1456  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
lac operator
1504 .. 1528  =  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
1504 .. 1528  =  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).
TEV site
232 .. 252  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
232 .. 252  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
263 .. 283  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
263 .. 283  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
1529 .. 1547  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1529 .. 1547  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
208 .. 225  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
208 .. 225  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
1468 .. 1473  =  6 bp
ribosome binding site
RBS
1468 .. 1473  =  6 bp
ribosome binding site
ATG
1457 .. 1459  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1457 .. 1459  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
tet operator
1846 .. 1864  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1846 .. 1864  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1871 .. 1889  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1871 .. 1889  =  19 bp
bacterial operator O2 for the tetR and tetA genes
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