pMCentr3

Plasmid with a kanamycin resistance marker and a TVMV cleavage site, for creating a Gateway® entry vector by ligation-independent cloning (LIC).

Sequence Author: Midwest Center for Structural Genomics

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PciI (2580) DrdI (2478) BssSI - BssSαI (2407) BciVI (2382) BsiHKAI (2270) ApaLI (2266) AlwNI (2171) AcuI - Eco57MI (2038) PflMI (1668) Bpu10I (1422) BsmBI - Esp3I (1421) AsiSI - PvuI (1405) BsrFI (1359) NspI (2584) BspQI - SapI (111) BsaHI (327) AclI (364) BbsI (437) HincII - HpaI (501) AhdI (549) AflII (554) AvaI - BsoBI (560) PspOMI (563) EcoO109I (564) ApaI (567) MslI (618) BsaI (665) XcmI (692) SspI (694) EcoRV (823) M13 rev NruI (1062) EcoNI (1317) pMCentr3 2586 bp
PciI  (2580)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (2478)
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.
BssSI  (2407)
1 site
C A C G A G G T G C T C
BssSαI  (2407)
1 site
C A C G A G G T G C T C
BciVI  (2382)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
BsiHKAI  (2270)
1 site
G W G C W C C W C G W G

Sticky ends from different BsiHKAI sites may not be compatible.
ApaLI  (2266)
1 site
G T G C A C C A C G T G
AlwNI  (2171)
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.
AcuI  (2038)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Cleavage may be enhanced when more than one copy of the AcuI recognition sequence is present.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
Eco57MI  (2038)
1 site
C T G R A G ( N ) 14 N N G A C Y T C ( N ) 14

Sticky ends from different Eco57MI sites may not be compatible.
After cleavage, Eco57MI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
PflMI  (1668)
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.
Bpu10I  (1422)
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.
BsmBI  (1421)
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.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (1421)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I sites may not be compatible.
AsiSI  (1405)
1 site
G C G A T C G C C G C T A G C G
PvuI  (1405)
1 site
C G A T C G G C T A G C
BsrFI  (1359)
1 site
R C C G G Y Y G G C C R

Cleavage may be enhanced when more than one copy of the BsrFI recognition sequence is present.
After cleavage, BsrFI can remain bound to DNA and alter its electrophoretic mobility.
NspI  (2584)
1 site
R C A T G Y Y G T A C R
BspQI  (111)
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  (111)
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.
BsaHI  (327)
1 site
G R C G Y C C Y G C R G

BsaHI is typically used at 37°C, but is even more active at 60°C.
AclI  (364)
1 site
A A C G T T T T G C A A
BbsI  (437)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
HincII  (501)
1 site
G T Y R A C C A R Y T G
HpaI  (501)
1 site
G T T A A C C A A T T G
AhdI  (549)
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.
AflII  (554)
1 site
C T T A A G G A A T T C
AvaI  (560)
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  (560)
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.
PspOMI  (563)
1 site
G G G C C C C C C G G G
EcoO109I  (564)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
ApaI  (567)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
MslI  (618)
1 site
C A Y N N N N R T G G T R N N N N Y A C
BsaI  (665)
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.
XcmI  (692)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
SspI  (694)
1 site
A A T A T T T T A T A A
EcoRV  (823)
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.
NruI  (1062)
1 site
T C G C G A A G C G C T
EcoNI  (1317)
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.
KanR
980 .. 1789  =  810 bp
269 amino acids  =  30.8 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
980 .. 1789  =  810 bp
269 amino acids  =  30.8 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
ori
1936 .. 2524  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1936 .. 2524  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
attL1
569 .. 668  =  100 bp
recombination site for the Gateway® LR reaction
attL1
569 .. 668  =  100 bp
recombination site for the Gateway® LR reaction
attL2
711 .. 810  =  100 bp
recombination site for the Gateway® LR reaction
attL2
711 .. 810  =  100 bp
recombination site for the Gateway® LR reaction
rrnB T1 terminator
387 .. 473  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
387 .. 473  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T2 terminator
268 .. 295  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
rrnB T2 terminator
268 .. 295  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
TVMV site
671 .. 691  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TVMV site
671 .. 691  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
T7 promoter
828 .. 846  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
828 .. 846  =  19 bp
promoter for bacteriophage T7 RNA polymerase
M13 fwd
537 .. 553  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
537 .. 553  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
851 .. 867  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
851 .. 867  =  17 bp
common sequencing primer, one of multiple similar variants
ORF:  980 .. 1789  =  810 bp
ORF:  269 amino acids  =  30.8 kDa
ORF:  394 .. 738  =  345 bp
ORF:  114 amino acids  =  12.8 kDa
ORF:  1093 .. 1332  =  240 bp
ORF:  79 amino acids  =  8.4 kDa
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