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pMCSG54 (linearized)

Linearized compact bacterial vector for ligation-independent cloning (LIC), with a 6xHis-FLAG®-TEV leader. 

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pMCSG54 (linearized) Sequence and MappMCSG54 (linearized).dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 StyI (4140) BspEI (4085) XmnI (3507) ScaI (3388) PvuI (3278) PstI (3153) FspI (3130) BglI (3028) BsaI (2969) AhdI (2908) AlwNI (2431) DrdI (2123) PciI (2015) BspQI - SapI (1899) BstZ17I (1786) SpeI (1764) BlpI (4163) 6xHis AvaI - BsoBI - PaeR7I - PspXI - XhoI (4241) BmeT110I (4242) EagI - NotI (4249) HindIII (4256) SalI (4262) Eco53kI (4271) SacI (4273) EcoRI (4275) BamHI (4281) End (4287) Start (0) TEV site Acc65I (25) KpnI (29) FLAG PsiI (49) BglII (56) 6xHis ATG NdeI (92) RBS XbaI (130) lac operator T7 promoter BspDI * - ClaI * (199) SgrAI (241) AfeI (327) BtgI (359) SphI (397) EcoNI (457) PfoI (489) PflMI (504) BstAPI (605) MluI (922) BclI * (936) BstEII (1103) PspOMI (1129) ApaI (1133) BssHII (1333) EcoRV (1372) HpaI (1428) BsmBI (1537) pMCSG54 4287 bp
StyI  (4140)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
BspEI  (4085)
1 site
T C C G G A A G G C C T
XmnI  (3507)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (3388)
1 site
A G T A C T T C A T G A
PvuI  (3278)
1 site
C G A T C G G C T A G C
PstI  (3153)
1 site
C T G C A G G A C G T C
FspI  (3130)
1 site
T G C G C A A C G C G T
BglI  (3028)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
BsaI  (2969)
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  (2908)
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  (2431)
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.
DrdI  (2123)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
PciI  (2015)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (1899)
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  (1899)
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  (1786)
1 site
G T A T A C C A T A T G
SpeI  (1764)
1 site
A C T A G T T G A T C A
BlpI  (4163)
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.
AvaI  (4241)
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  (4241)
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  (4241)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4241)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4241)
1 site
C T C G A G G A G C T C
BmeT110I  (4242)
1 site
C Y C G R G G R G C Y C
EagI  (4249)
1 site
C G G C C G G C C G G C
NotI  (4249)
1 site
G C G G C C G C C G C C G G C G
HindIII  (4256)
1 site
A A G C T T T T C G A A
SalI  (4262)
1 site
G T C G A C C A G C T G
Eco53kI  (4271)
1 site
G A G C T C C T C G A G
SacI  (4273)
1 site
G A G C T C C T C G A G
EcoRI  (4275)
1 site
G A A T T C C T T A A G
BamHI  (4281)
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  (4287)
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.
AfeI  (327)
1 site
A G C G C T T C G C G A
BtgI  (359)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
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.
PfoI  (489)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI 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
BsmBI  (1537)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI 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
2835 .. 3695  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   2835 .. 3626  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2835 .. 3695  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   3627 .. 3695  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2835 .. 3695  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2076 .. 2664  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2076 .. 2664  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
AmpR promoter
3696 .. 3799  =  104 bp
AmpR promoter
3696 .. 3799  =  104 bp
lacI promoter
494 .. 571  =  78 bp
lacI promoter
494 .. 571  =  78 bp
T7 terminator
4109 .. 4156  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
4109 .. 4156  =  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
4223 .. 4240  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4223 .. 4240  =  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
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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