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Plasmid Files

pMCSG64 (linearized)

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

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pMCSG64 (linearized) Sequence and MappMCSG64 (linearized).dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 T7 terminator ScaI (4396) PvuI (4286) PstI (4161) BsaI (3977) AhdI (3916) AlwNI (3439) PciI (3023) BspQI - SapI (2907) BstZ17I (2794) BsaAI (2775) PflFI - Tth111I (2768) Bpu10I (2129) StyI (5148) BlpI (5171) PaeR7I - PspXI - XhoI (5249) EagI - NotI (5257) HindIII (5264) SalI (5270) Eco53kI (5279) SacI (5281) EcoRI (5283) BamHI (5289) TspMI - XmaI (5323) SmaI (5325) End (5351) Start (0) TEV site Acc65I (25) KpnI (29) PsiI (49) BglII (56) 6xHis ATG NdeI (92) RBS XbaI (130) lac operator T7 promoter BspDI * - ClaI * (199) SgrAI (241) SphI (397) EcoNI (457) PflMI (504) BstAPI (605) MluI (922) BclI * (936) BstEII (1103) PspOMI (1129) ApaI (1133) BssHII (1333) EcoRV (1372) HpaI (1428) PshAI (1767) FspAI (2004) PpuMI (2029) pMCSG64 5351 bp
ScaI  (4396)
1 site
A G T A C T T C A T G A
PvuI  (4286)
1 site
C G A T C G G C T A G C
PstI  (4161)
1 site
C T G C A G G A C G T C
BsaI  (3977)
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  (3916)
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  (3439)
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  (3023)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2907)
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  (2907)
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  (2794)
1 site
G T A T A C C A T A T G
BsaAI  (2775)
1 site
Y A C G T R R T G C A Y
PflFI  (2768)
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  (2768)
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  (2129)
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  (5148)
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  (5171)
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  (5249)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5249)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5249)
1 site
C T C G A G G A G C T C
EagI  (5257)
1 site
C G G C C G G C C G G C
NotI  (5257)
1 site
G C G G C C G C C G C C G G C G
HindIII  (5264)
1 site
A A G C T T T T C G A A
SalI  (5270)
1 site
G T C G A C C A G C T G
Eco53kI  (5279)
1 site
G A G C T C C T C G A G
SacI  (5281)
1 site
G A G C T C C T C G A G
EcoRI  (5283)
1 site
G A A T T C C T T A A G
BamHI  (5289)
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  (5323)
1 site
C C C G G G G G G C C C
XmaI  (5323)
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  (5325)
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  (5351)
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
PsiI  (49)
1 site
T T A T A A A A T A T T
BglII  (56)
1 site
A G A T C T T C T A G A
NdeI  (92)
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  (130)
1 site
T C T A G A A G A T C T
BspDI  (199)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (199)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (241)
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  (397)
1 site
G C A T G C C G T A C G
EcoNI  (457)
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  (504)
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  (605)
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  (922)
1 site
A C G C G T T G C G C A
BclI  (936)
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  (1103)
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  (1129)
1 site
G G G C C C C C C G G G
ApaI  (1133)
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  (1333)
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  (1372)
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  (1428)
1 site
G T T A A C C A A T T G
PshAI  (1767)
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  (2004)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (2029)
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
572 .. 1654  =  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
572 .. 1654  =  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
3843 .. 4703  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   3843 .. 4634  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3843 .. 4703  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   4635 .. 4703  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3843 .. 4703  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3084 .. 3672  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3084 .. 3672  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
2463 .. 2654  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2463 .. 2654  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
4704 .. 4807  =  104 bp
AmpR promoter
4704 .. 4807  =  104 bp
lacI promoter
494 .. 571  =  78 bp
lacI promoter
494 .. 571  =  78 bp
T7 terminator
5117 .. 5164  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
5117 .. 5164  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
138 .. 162  =  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
138 .. 162  =  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).
FLAG
31 .. 54  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
31 .. 54  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
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
163 .. 181  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
163 .. 181  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
73 .. 90  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
73 .. 90  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5231 .. 5248  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5231 .. 5248  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
102 .. 107  =  6 bp
ribosome binding site
RBS
102 .. 107  =  6 bp
ribosome binding site
RBS
5334 .. 5339  =  6 bp
ribosome binding site
RBS
5334 .. 5339  =  6 bp
ribosome binding site
ATG
91 .. 93  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
91 .. 93  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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