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Plasmid Files

pMCSG25

Bacterial expression vector encoding an aspartic endopeptidase fused to a 6xHis-TEV leader.

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pMCSG25 Sequence and MappMCSG25.dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 ScaI (4969) PvuI (4859) PstI (4734) BsaI (4550) AhdI (4489) AlwNI (4012) PciI (3596) BspQI - SapI (3480) BstZ17I (3367) BsaAI (3348) PflFI - Tth111I (3341) T7 terminator 6xHis AvaI - BsoBI - PaeR7I - PspXI - XhoI (158) BmeT110I (159) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) SspI (219) Acc65I (244) KpnI (248) BglII (251) 6xHis SpeI (404) PsiI (635) RBS XbaI (703) lac operator T7 promoter SgrAI (814) SphI (970) EcoNI (1030) lacI promoter BstAPI (1178) MluI (1495) PspOMI (1702) ApaI (1706) BssHII (1906) HpaI (2001) PshAI (2340) FspAI (2577) PpuMI (2602) Bpu10I (2702) pMCSG25 5664 bp
ScaI  (4969)
1 site
A G T A C T T C A T G A
PvuI  (4859)
1 site
C G A T C G G C T A G C
PstI  (4734)
1 site
C T G C A G G A C G T C
BsaI  (4550)
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  (4489)
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.
AlwNI  (4012)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
PciI  (3596)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3480)
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  (3480)
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  (3367)
1 site
G T A T A C C A T A T G
BsaAI  (3348)
1 site
Y A C G T R R T G C A Y
PflFI  (3341)
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  (3341)
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.
AvaI  (158)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (158)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
PaeR7I  (158)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (158)
1 site
V C T C G A G B B G A G C T C V
XhoI  (158)
1 site
C T C G A G G A G C T C
BmeT110I  (159)
1 site
C Y C G R G G R G C Y C
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
HindIII  (173)
1 site
A A G C T T T T C G A A
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
EcoRI  (192)
1 site
G A A T T C C T T A 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
SpeI  (404)
1 site
A C T A G T T G A T C A
PsiI  (635)
1 site
T T A T A A A A T A T T
XbaI  (703)
1 site
T C T A G A A G A T C T
SgrAI  (814)
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  (970)
1 site
G C A T G C C G T A C G
EcoNI  (1030)
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.
BstAPI  (1178)
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.
MluI  (1495)
1 site
A C G C G T T G C G C A
PspOMI  (1702)
1 site
G G G C C C C C C G G G
ApaI  (1706)
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  (1906)
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.
HpaI  (2001)
1 site
G T T A A C C A A T T G
PshAI  (2340)
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  (2577)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (2602)
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.
Bpu10I  (2702)
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.
lacI
1145 .. 2227  =  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
1145 .. 2227  =  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
4416 .. 5276  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4416 .. 5207  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4416 .. 5276  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5208 .. 5276  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4416 .. 5276  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3657 .. 4245  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3657 .. 4245  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
aspartic endopeptidase
292 .. 666  =  375 bp
125 amino acids  =  13.4 kDa
Product: self-cleaving aspartic endopeptidase
SO1698 from Shewanella oneidensis
aspartic endopeptidase
292 .. 666  =  375 bp
125 amino acids  =  13.4 kDa
Product: self-cleaving aspartic endopeptidase
SO1698 from Shewanella oneidensis
rop
3036 .. 3227  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
3036 .. 3227  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
5277 .. 5380  =  104 bp
AmpR promoter
5277 .. 5380  =  104 bp
lacI promoter
1067 .. 1144  =  78 bp
lacI promoter
1067 .. 1144  =  78 bp
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
711 .. 735  =  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
711 .. 735  =  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
736 .. 754  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
736 .. 754  =  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
675 .. 680  =  6 bp
ribosome binding site
RBS
675 .. 680  =  6 bp
ribosome binding site
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