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Plasmid Files

pMCSG38

Bacterial expression vector with a 6xHis-TEV leader and a Tet-inducible TVMV protease cassette. See also pMCSG38C.

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pMCSG38 Sequence and MappMCSG38.dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 ScaI (5618) PvuI (5508) BsaI (5199) AhdI (5138) PciI (4245) BspQI - SapI (4129) BstZ17I (4016) BsaAI (3997) PflFI - Tth111I (3990) Bpu10I (3351) PpuMI (3251) BlpI (80) 6xHis EagI - NotI (166) SalI (179) Eco53kI (188) SacI (190) BamHI (198) SspI (219) Acc65I (244) KpnI (248) BglII (251) 6xHis ATG RBS lac operator BspDI * - ClaI * (394) SgrAI (436) SphI (592) NcoI (712) SacII (854) SpeI (967) DraIII (1043) AgeI (1204) NsiI (1255) BseRI (1375) ZraI (1611) AatII (1613) SphI (1619) EcoNI (1679) PflMI (1726) BstAPI (1827) BclI * (2158) BstEII (2325) PspOMI (2351) ApaI (2355) BssHII (2555) EcoRV (2594) HpaI (2650) PshAI (2989) FspAI (3226) pMCSG38 6313 bp
ScaI  (5618)
1 site
A G T A C T T C A T G A
PvuI  (5508)
1 site
C G A T C G G C T A G C
BsaI  (5199)
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.
AhdI  (5138)
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  (4245)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4129)
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  (4129)
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  (4016)
1 site
G T A T A C C A T A T G
BsaAI  (3997)
1 site
Y A C G T R R T G C A Y
PflFI  (3990)
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  (3990)
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  (3351)
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  (3251)
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.
BlpI  (80)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
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.
SspI  (219)
1 site
A A T A T T T T A T A A
Acc65I  (244)
1 site
G G T A C C C C A T G G
KpnI  (248)
1 site
G G T A C C C C A T G G
BglII  (251)
1 site
A G A T C T T C T A G A
BspDI  (394)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (394)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (436)
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  (592)
2 sites
G C A T G C C G T A C G
NcoI  (712)
1 site
C C A T G G G G T A C C
SacII  (854)
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.
SpeI  (967)
1 site
A C T A G T T G A T C A
DraIII  (1043)
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.
AgeI  (1204)
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.
NsiI  (1255)
1 site
A T G C A T T A C G T A
BseRI  (1375)
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.
ZraI  (1611)
1 site
G A C G T C C T G C A G
AatII  (1613)
1 site
G A C G T C C T G C A G
SphI  (1619)
2 sites
G C A T G C C G T A C G
EcoNI  (1679)
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  (1726)
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.
BstAPI  (1827)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
BclI  (2158)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
BstEII  (2325)
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  (2351)
1 site
G G G C C C C C C G G G
ApaI  (2355)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BssHII  (2555)
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.
EcoRV  (2594)
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  (2650)
1 site
G T T A A C C A A T T G
PshAI  (2989)
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.
FspAI  (3226)
1 site
R T G C G C A Y Y A C G C G T R
lacI
1794 .. 2876  =  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
1794 .. 2876  =  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
5065 .. 5925  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   5065 .. 5856  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5065 .. 5925  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5857 .. 5925  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5065 .. 5925  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TVMV protease
726 .. 1436  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease
(Nallamsetty et al., 2004)
TVMV protease
726 .. 1436  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease
(Nallamsetty et al., 2004)
ori
4306 .. 4894  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4306 .. 4894  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
3685 .. 3876  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
3685 .. 3876  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
5926 .. 6029  =  104 bp
AmpR promoter
5926 .. 6029  =  104 bp
rrnB T1 terminator
607 .. 693  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
607 .. 693  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
lacI promoter
1716 .. 1793  =  78 bp
lacI promoter
1716 .. 1793  =  78 bp
PLtetO-1 promoter
1460 .. 1533  =  74 bp
   Segment 5:  
   1460 .. 1485  =  26 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1460 .. 1533  =  74 bp
   Segment 4:  -10  
   1486 .. 1491  =  6 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1460 .. 1533  =  74 bp
   Segment 3:  
   1492 .. 1508  =  17 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1460 .. 1533  =  74 bp
   Segment 2:  -35  
   1509 .. 1514  =  6 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1460 .. 1533  =  74 bp
   Segment 1:  
   1515 .. 1533  =  19 bp
modified phage lambda PL promoter with tet
operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1460 .. 1533  =  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
lac operator
333 .. 357  =  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
333 .. 357  =  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
223 .. 243  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
223 .. 243  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
358 .. 376  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
358 .. 376  =  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
268 .. 285  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
268 .. 285  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
297 .. 302  =  6 bp
ribosome binding site
RBS
297 .. 302  =  6 bp
ribosome binding site
ATG
286 .. 288  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
286 .. 288  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
tet operator
1490 .. 1508  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1490 .. 1508  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1515 .. 1533  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1515 .. 1533  =  19 bp
bacterial operator O2 for the tetR and tetA genes
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