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.

Sequence Author: Midwest Center for Structural Genomics

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StyI (5148) 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) 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
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.
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

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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.
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

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
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
2 segments
   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
2 segments
   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
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
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
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
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
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).
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
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
ORF:  1 .. 822  =  822 bp
ORF:  273 amino acids  =  29.1 kDa  (no start codon)
ORF:  1411 .. 1674  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  3973 .. 4239  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  695 .. 1654  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  1710 .. 2066  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  2430 .. 2654  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  270 .. 533  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  1437 .. 1688  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  3843 .. 4703  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2063 .. 2431  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  145 .. 396  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
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