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Plasmid Files

pMCSG67

Bacterial vector for expressing a protein with a 6xHis-Strep-Tag® II-TEV leader plus a second untagged protein.

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pMCSG67 Sequence and MappMCSG67.dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 EagI - NotI (166) PaeR7I - PspXI - XhoI (158) BlpI (80) StyI (57) T7 terminator ScaI (4671) PvuI (4561) PstI (4436) AhdI (4191) AlwNI (3714) PciI (3298) BspQI - SapI (3182) BstZ17I (3069) BsaAI (3050) PflFI - Tth111I (3043) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) TspMI - XmaI (232) SmaI (234) RBS SspI (275) Acc65I (300) KpnI (304) BstBI (310) Strep-Tag II BglII (331) 6xHis ATG NdeI (367) RBS XbaI (405) lac operator T7 promoter BspDI * - ClaI * (474) SgrAI (516) SphI (672) EcoNI (732) PflMI (779) BstAPI (880) MluI (1197) BclI * (1211) BstEII (1378) PspOMI (1404) ApaI (1408) BssHII (1608) EcoRV (1647) HpaI (1703) PshAI (2042) FspAI (2279) PpuMI (2304) Bpu10I (2404) pMCSG67 5366 bp
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
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
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.
StyI  (57)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
ScaI  (4671)
1 site
A G T A C T T C A T G A
PvuI  (4561)
1 site
C G A T C G G C T A G C
PstI  (4436)
1 site
C T G C A G G A C G T C
AhdI  (4191)
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  (3714)
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  (3298)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3182)
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  (3182)
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  (3069)
1 site
G T A T A C C A T A T G
BsaAI  (3050)
1 site
Y A C G T R R T G C A Y
PflFI  (3043)
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  (3043)
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.
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.
TspMI  (232)
1 site
C C C G G G G G G C C C
XmaI  (232)
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  (234)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SspI  (275)
1 site
A A T A T T T T A T A A
Acc65I  (300)
1 site
G G T A C C C C A T G G
KpnI  (304)
1 site
G G T A C C C C A T G G
BstBI  (310)
1 site
T T C G A A A A G C T T
BglII  (331)
1 site
A G A T C T T C T A G A
NdeI  (367)
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  (405)
1 site
T C T A G A A G A T C T
BspDI  (474)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (474)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (516)
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  (672)
1 site
G C A T G C C G T A C G
EcoNI  (732)
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  (779)
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  (880)
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  (1197)
1 site
A C G C G T T G C G C A
BclI  (1211)
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  (1378)
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  (1404)
1 site
G G G C C C C C C G G G
ApaI  (1408)
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  (1608)
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  (1647)
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  (1703)
1 site
G T T A A C C A A T T G
PshAI  (2042)
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  (2279)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (2304)
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  (2404)
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
847 .. 1929  =  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
847 .. 1929  =  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
4118 .. 4978  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4118 .. 4909  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4118 .. 4978  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   4910 .. 4978  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4118 .. 4978  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3359 .. 3947  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3359 .. 3947  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
2738 .. 2929  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2738 .. 2929  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
4979 .. 5082  =  104 bp
AmpR promoter
4979 .. 5082  =  104 bp
lacI promoter
769 .. 846  =  78 bp
lacI promoter
769 .. 846  =  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
413 .. 437  =  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
413 .. 437  =  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).
Strep-Tag II
306 .. 329  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin®, an
engineered form of streptavidin
Strep-Tag II
306 .. 329  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin®, an
engineered form of streptavidin
TEV site
279 .. 299  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
279 .. 299  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
438 .. 456  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
438 .. 456  =  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
348 .. 365  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
348 .. 365  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
243 .. 248  =  6 bp
ribosome binding site
RBS
243 .. 248  =  6 bp
ribosome binding site
RBS
377 .. 382  =  6 bp
ribosome binding site
RBS
377 .. 382  =  6 bp
ribosome binding site
ATG
366 .. 368  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
366 .. 368  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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