pKF 19k-2

Bacterial cloning and site-directed mutagenesis vector with a kanamycin resistance gene containing amber stop codons. The MCS is reversed in pKF 18k-2.

Sequence Author: TaKaRa

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NdeI (2203) lac operator BsrBI (2175) DrdI (1992) BciVI (1896) HaeII (1854) PspFI (1794) BseYI (1790) BaeGI - Bme1580I (1784) ApaLI (1780) AlwNI (1685) AcuI - Eco57MI (1552) StuI * (1352) DraIII (1227) BsrDI (1203) BspHI (1171) HindIII (18) BfuAI - BspMI (23) NspI - SphI (28) PstI - SbfI (34) SalI (36) AccI (37) HincII (38) XbaI (42) BamHI (48) AvaI - BsoBI - TspMI - XmaI (53) SmaI (55) Acc65I (57) KpnI (61) Eco53kI (65) SacI (67) EcoRI (69) EaeI (77) BmrI (111) PvuII (161) FspI (211) BglI (221) PflMI (433) TaqII (441) AseI (492) Bpu10I - BsmBI - Esp3I (673) AsiSI (695) BsrFI (735) SspI (768) EcoNI (780) NruI (1036) pKF 19k-2 2204 bp
NdeI  (2203)
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.
BsrBI  (2175)
1 site
C C G C T C G G C G A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BsrBI will not always regenerate a BsrBI site.
BsrBI is typically used at 37°C, but can be used at temperatures up to 50°C.
DrdI  (1992)
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.
BciVI  (1896)
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.
HaeII  (1854)
1 site
R G C G C Y Y C G C G R
PspFI  (1794)
1 site
C C C A G C G G G T C G
BseYI  (1790)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
BaeGI  (1784)
1 site
G K G C M C C M C G K G

Sticky ends from different BaeGI sites may not be compatible.
Bme1580I  (1784)
1 site
G K G C M C C M C G K G

Sticky ends from different Bme1580I sites may not be compatible.
ApaLI  (1780)
1 site
G T G C A C C A C G T G
AlwNI  (1685)
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  (1552)
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  (1552)
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).
StuI  (1352)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
DraIII  (1227)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
BsrDI  (1203)
1 site
G C A A T G N N C G T T A C

Sticky ends from different BsrDI sites may not be compatible.
BspHI  (1171)
1 site
T C A T G A A G T A C T
HindIII  (18)
1 site
A A G C T T T T C G A A
BfuAI  (23)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (23)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
NspI  (28)
1 site
R C A T G Y Y G T A C R
SphI  (28)
1 site
G C A T G C C G T A C G
PstI  (34)
1 site
C T G C A G G A C G T C
SbfI  (34)
1 site
C C T G C A G G G G A C G T C C
SalI  (36)
1 site
G T C G A C C A G C T G
AccI  (37)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
HincII  (38)
1 site
G T Y R A C C A R Y T G
XbaI  (42)
1 site
T C T A G A A G A T C T
BamHI  (48)
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.
AvaI  (53)
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  (53)
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.
TspMI  (53)
1 site
C C C G G G G G G C C C
XmaI  (53)
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  (55)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
Acc65I  (57)
1 site
G G T A C C C C A T G G
KpnI  (61)
1 site
G G T A C C C C A T G G
Eco53kI  (65)
1 site
G A G C T C C T C G A G
SacI  (67)
1 site
G A G C T C C T C G A G
EcoRI  (69)
1 site
G A A T T C C T T A A G
EaeI  (77)
1 site
Y G G C C R R C C G G Y
BmrI  (111)
1 site
A C T G G G ( N ) 4 N T G A C C C ( N ) 4

The 1-base overhangs produced by BmrI may be hard to ligate.
Sticky ends from different BmrI sites may not be compatible.
Unlike most restriction enzymes, BmrI can cleave DNA in the absence of magnesium.
PvuII  (161)
1 site
C A G C T G G T C G A C
FspI  (211)
1 site
T G C G C A A C G C G T
BglI  (221)
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.
PflMI  (433)
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.
TaqII  (441)
1 site
G A C C G A ( N ) 9 N N C T G G C T ( N ) 9

Sticky ends from different TaqII sites may not be compatible.
AseI  (492)
1 site
A T T A A T T A A T T A
Bpu10I  (673)
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  (673)
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  (673)
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  (695)
1 site
G C G A T C G C C G C T A G C G
BsrFI  (735)
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.
SspI  (768)
1 site
A A T A T T T T A T A A
EcoNI  (780)
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.
NruI  (1036)
1 site
T C G C G A A G C G C T
KanR (with amber stop codons)
310 .. 1125  =  816 bp
272 codons
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR (with amber stop codons)
310 .. 1125  =  816 bp
272 codons
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
ori
1450 .. 2038  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1450 .. 2038  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
lacZα
1 .. 309  =  309 bp
102 amino acids  =  11.4 kDa
Product: LacZα fragment of β-galactosidase
lacZα
1 .. 309  =  309 bp
102 amino acids  =  11.4 kDa
Product: LacZα fragment of β-galactosidase
lac promoter
2131 .. 2161  =  31 bp
3 segments
   Segment 1:  -35  
   2131 .. 2136  =  6 bp
promoter for the E. coli lac operon
lac promoter
2131 .. 2161  =  31 bp
3 segments
   Segment 2:  
   2137 .. 2154  =  18 bp
promoter for the E. coli lac operon
lac promoter
2131 .. 2161  =  31 bp
3 segments
   Segment 3:  -10  
   2155 .. 2161  =  7 bp
promoter for the E. coli lac operon
lac promoter
2131 .. 2161  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
2169 .. 2185  =  17 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
2169 .. 2185  =  17 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).
MCS
18 .. 74  =  57 bp
pUC18/19 multiple cloning site
MCS
18 .. 74  =  57 bp
pUC18/19 multiple cloning site
M13 fwd
75 .. 91  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
75 .. 91  =  17 bp
common sequencing primer, one of multiple similar variants
ORF:  1 .. 309  =  309 bp
ORF:  102 amino acids  =  11.4 kDa
ORF:  310 .. 660  =  351 bp
ORF:  116 amino acids  =  13.4 kDa
ORF:  673 .. 1125  =  453 bp
ORF:  150 amino acids  =  17.0 kDa
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Download pKF 19k-2.dna file

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