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pMCSG49 (linearized)

Linearized compact bacterial vector for ligation-independent cloning (LIC), with a 6xHis-TEV leader. Derived from pMCSG7 by removing the rop gene.

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pMCSG49 (linearized).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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BspEI (4061) XmnI (3483) ScaI (3364) PvuI (3254) PstI (3129) FspI (3106) BglI (3004) BsaI (2945) AhdI (2884) AlwNI (2407) DrdI (2099) PciI (1991) BspQI - SapI (1875) StyI (4116) BlpI (4139) AvaI - BsoBI - PaeR7I - PspXI - XhoI (4217) BmeT110I (4218) EagI - NotI (4225) HindIII (4232) SalI (4238) Eco53kI (4247) SacI (4249) EcoRI (4251) BamHI (4257) End (4263) Start (0) Acc65I (25) KpnI (29) BglII (32) NdeI (68) XbaI (106) BspDI * - ClaI * (175) SgrAI (217) AfeI (303) BtgI (335) SphI (373) EcoNI (433) PfoI (465) PflMI (480) BstAPI (581) MluI (898) BclI * (912) BstEII (1079) PspOMI (1105) ApaI (1109) BssHII (1309) EcoRV (1348) HpaI (1404) BsmBI (1513) SpeI (1740) BstZ17I (1762) pMCSG49 4263 bp
BspEI  (4061)
1 site
T C C G G A A G G C C T
XmnI  (3483)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (3364)
1 site
A G T A C T T C A T G A
PvuI  (3254)
1 site
C G A T C G G C T A G C
PstI  (3129)
1 site
C T G C A G G A C G T C
FspI  (3106)
1 site
T G C G C A A C G C G T
BglI  (3004)
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  (2945)
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  (2884)
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  (2407)
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  (2099)
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  (1991)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (1875)
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  (1875)
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.
StyI  (4116)
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  (4139)
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  (4217)
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  (4217)
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  (4217)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4217)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4217)
1 site
C T C G A G G A G C T C
BmeT110I  (4218)
1 site
C Y C G R G G R G C Y C
EagI  (4225)
1 site
C G G C C G G C C G G C
NotI  (4225)
1 site
G C G G C C G C C G C C G G C G
HindIII  (4232)
1 site
A A G C T T T T C G A A
SalI  (4238)
1 site
G T C G A C C A G C T G
Eco53kI  (4247)
1 site
G A G C T C C T C G A G
SacI  (4249)
1 site
G A G C T C C T C G A G
EcoRI  (4251)
1 site
G A A T T C C T T A A G
BamHI  (4257)
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  (4263)
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.
AfeI  (303)
1 site
A G C G C T T C G C G A
BtgI  (335)
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  (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.
PfoI  (465)
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  (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
BsmBI  (1513)
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.
SpeI  (1740)
1 site
A C T A G T T G A T C A
BstZ17I  (1762)
1 site
G T A T A C C A T A T G
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
2811 .. 3671  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   2811 .. 3602  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2811 .. 3671  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   3603 .. 3671  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2811 .. 3671  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2052 .. 2640  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2052 .. 2640  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
3672 .. 3775  =  104 bp
AmpR promoter
3672 .. 3775  =  104 bp
lacI promoter
470 .. 547  =  78 bp
lacI promoter
470 .. 547  =  78 bp
T7 terminator
4085 .. 4132  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
4085 .. 4132  =  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
4199 .. 4216  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4199 .. 4216  =  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
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
ORF:  1 .. 798  =  798 bp
ORF:  265 amino acids  =  28.2 kDa  (no start codon)
ORF:  1387 .. 1650  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  2941 .. 3207  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  671 .. 1630  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  121 .. 372  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  246 .. 509  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  1413 .. 1664  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  2811 .. 3671  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
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