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Plasmid Files

pMCSG19 (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC), with an MBP-TVMV-6xHis-TEV leader.

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pMCSG19 (linearized) Sequence and MappMCSG19 (linearized).dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 AvaI - BsoBI - PaeR7I - PspXI - XhoI (6380) ScaI (5527) PstI (5292) AhdI (5047) PciI (4154) BspQI - SapI (4038) BstZ17I (3925) PflFI - Tth111I (3899) Bpu10I (3260) PpuMI (3160) FspAI (3135) PshAI (2898) BmeT110I (6381) EagI - NotI (6388) HindIII (6395) SalI (6401) Eco53kI (6410) SacI (6412) EcoRI (6414) BamHI (6420) End (6426) Start (0) Acc65I (25) KpnI (29) 6xHis TVMV site BsrGI (102) NcoI (258) BsmI (605) BmgBI (681) BglII (860) PsiI (871) BsiWI (929) NdeI (1223) RBS XbaI (1261) T7 promoter BspDI * - ClaI * (1330) SgrAI (1372) SphI (1528) EcoNI (1588) PflMI (1635) MluI (2053) BstEII (2234) PspOMI (2260) ApaI (2264) EcoRV (2503) HpaI (2559) pMCSG19 6426 bp
AvaI  (6380)
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  (6380)
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  (6380)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (6380)
1 site
V C T C G A G B B G A G C T C V
XhoI  (6380)
1 site
C T C G A G G A G C T C
ScaI  (5527)
1 site
A G T A C T T C A T G A
PstI  (5292)
1 site
C T G C A G G A C G T C
AhdI  (5047)
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  (4154)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4038)
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  (4038)
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  (3925)
1 site
G T A T A C C A T A T G
PflFI  (3899)
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  (3899)
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  (3260)
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  (3160)
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  (3135)
1 site
R T G C G C A Y Y A C G C G T R
PshAI  (2898)
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.
BmeT110I  (6381)
1 site
C Y C G R G G R G C Y C
EagI  (6388)
1 site
C G G C C G G C C G G C
NotI  (6388)
1 site
G C G G C C G C C G C C G G C G
HindIII  (6395)
1 site
A A G C T T T T C G A A
SalI  (6401)
1 site
G T C G A C C A G C T G
Eco53kI  (6410)
1 site
G A G C T C C T C G A G
SacI  (6412)
1 site
G A G C T C C T C G A G
EcoRI  (6414)
1 site
G A A T T C C T T A A G
BamHI  (6420)
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.
End  (6426)
0 sites
Start  (0)
0 sites
Acc65I  (25)
1 site
G G T A C C C C A T G G
KpnI  (29)
1 site
G G T A C C C C A T G G
BsrGI  (102)
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.
NcoI  (258)
1 site
C C A T G G G G T A C C
BsmI  (605)
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  (681)
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.
BglII  (860)
1 site
A G A T C T T C T A G A
PsiI  (871)
1 site
T T A T A A A A T A T T
BsiWI  (929)
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.
NdeI  (1223)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
XbaI  (1261)
1 site
T C T A G A A G A T C T
BspDI  (1330)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1330)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (1372)
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  (1528)
1 site
G C A T G C C G T A C G
EcoNI  (1588)
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  (1635)
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.
MluI  (2053)
1 site
A C G C G T T G C G C A
BstEII  (2234)
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  (2260)
1 site
G G G C C C C C C G G G
ApaI  (2264)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
EcoRV  (2503)
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  (2559)
1 site
G T T A A C C A A T T G
MBP
124 .. 1224  =  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
124 .. 1224  =  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.
lacI
1703 .. 2785  =  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
1703 .. 2785  =  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
4974 .. 5834  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4974 .. 5765  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4974 .. 5834  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5766 .. 5834  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4974 .. 5834  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
4215 .. 4803  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4215 .. 4803  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
3594 .. 3785  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
3594 .. 3785  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
5835 .. 5938  =  104 bp
AmpR promoter
5835 .. 5938  =  104 bp
lacI promoter
1625 .. 1702  =  78 bp
lacI promoter
1625 .. 1702  =  78 bp
T7 terminator
6248 .. 6295  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
6248 .. 6295  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
1269 .. 1293  =  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
1269 .. 1293  =  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
4 .. 24  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) recognition and
cleavage site
TEV site
4 .. 24  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) recognition and
cleavage site
TVMV site
67 .. 87  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa
protease recognition and cleavage site
TVMV site
67 .. 87  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa
protease recognition and cleavage site
T7 promoter
1294 .. 1312  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1294 .. 1312  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
49 .. 66  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
49 .. 66  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
6362 .. 6379  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
6362 .. 6379  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
1233 .. 1238  =  6 bp
ribosome binding site
RBS
1233 .. 1238  =  6 bp
ribosome binding site
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