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Plasmid Files

pMCSG60 (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC), with a 6xHis-TEV leader plus a second LIC site for expressing an untagged protein.

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pMCSG60 (linearized) Sequence and MappMCSG60 (linearized).dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 T7 terminator ScaI (4372) PvuI (4262) PstI (4137) BsaI (3953) AhdI (3892) AlwNI (3415) PciI (2999) BspQI - SapI (2883) BstZ17I (2770) BsaAI (2751) PflFI - Tth111I (2744) Bpu10I (2105) StyI (5124) BlpI (5147) 6xHis PaeR7I - PspXI - XhoI (5225) EagI - NotI (5233) HindIII (5240) SalI (5246) Eco53kI (5255) SacI (5257) EcoRI (5259) BamHI (5265) TspMI - XmaI (5299) SmaI (5301) RBS End (5327) Start (0) Acc65I (25) KpnI (29) BglII (32) 6xHis ATG NdeI (68) RBS XbaI (106) T7 promoter BspDI * - ClaI * (175) SgrAI (217) SphI (373) EcoNI (433) PflMI (480) BstAPI (581) MluI (898) BclI * (912) BstEII (1079) PspOMI (1105) ApaI (1109) BssHII (1309) EcoRV (1348) HpaI (1404) PshAI (1743) FspAI (1980) PpuMI (2005) pMCSG60 5327 bp
ScaI  (4372)
1 site
A G T A C T T C A T G A
PvuI  (4262)
1 site
C G A T C G G C T A G C
PstI  (4137)
1 site
C T G C A G G A C G T C
BsaI  (3953)
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  (3892)
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  (3415)
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  (2999)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2883)
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  (2883)
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  (2770)
1 site
G T A T A C C A T A T G
BsaAI  (2751)
1 site
Y A C G T R R T G C A Y
PflFI  (2744)
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  (2744)
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  (2105)
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.
StyI  (5124)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
BlpI  (5147)
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.
PaeR7I  (5225)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5225)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5225)
1 site
C T C G A G G A G C T C
EagI  (5233)
1 site
C G G C C G G C C G G C
NotI  (5233)
1 site
G C G G C C G C C G C C G G C G
HindIII  (5240)
1 site
A A G C T T T T C G A A
SalI  (5246)
1 site
G T C G A C C A G C T G
Eco53kI  (5255)
1 site
G A G C T C C T C G A G
SacI  (5257)
1 site
G A G C T C C T C G A G
EcoRI  (5259)
1 site
G A A T T C C T T A A G
BamHI  (5265)
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  (5299)
1 site
C C C G G G G G G C C C
XmaI  (5299)
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  (5301)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
End  (5327)
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
BglII  (32)
1 site
A G A T C T T C T A G A
NdeI  (68)
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  (106)
1 site
T C T A G A A G A T C T
BspDI  (175)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (175)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (217)
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  (373)
1 site
G C A T G C C G T A C G
EcoNI  (433)
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  (480)
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  (581)
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  (898)
1 site
A C G C G T T G C G C A
BclI  (912)
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  (1079)
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  (1105)
1 site
G G G C C C C C C G G G
ApaI  (1109)
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  (1309)
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  (1348)
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  (1404)
1 site
G T T A A C C A A T T G
PshAI  (1743)
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  (1980)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (2005)
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.
lacI
548 .. 1630  =  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
548 .. 1630  =  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
3819 .. 4679  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   3819 .. 4610  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3819 .. 4679  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   4611 .. 4679  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3819 .. 4679  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3060 .. 3648  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3060 .. 3648  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
2439 .. 2630  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2439 .. 2630  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
4680 .. 4783  =  104 bp
AmpR promoter
4680 .. 4783  =  104 bp
lacI promoter
470 .. 547  =  78 bp
lacI promoter
470 .. 547  =  78 bp
T7 terminator
5093 .. 5140  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
5093 .. 5140  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
114 .. 138  =  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
114 .. 138  =  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) protease
recognition and cleavage site
TEV site
4 .. 24  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
139 .. 157  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
139 .. 157  =  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
5207 .. 5224  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5207 .. 5224  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
78 .. 83  =  6 bp
ribosome binding site
RBS
78 .. 83  =  6 bp
ribosome binding site
RBS
5310 .. 5315  =  6 bp
ribosome binding site
RBS
5310 .. 5315  =  6 bp
ribosome binding site
ATG
67 .. 69  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
67 .. 69  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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