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Plasmid Files

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.

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pKF 19k-2 Sequence and MappKF 19k-2.dna
Map and Sequence File   
Sequence Author:  TaKaRa
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 NdeI (2203) 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) BmeT110I (54) 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 (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

Efficient cleavage requires at least two copies of the AcuI
recognition sequence.
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

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
BmeT110I  (54)
1 site
C Y C G R G G R G C Y C
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

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
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.
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

Efficient cleavage requires at least two copies of the BsrFI
recognition sequence.
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
   Segment 1:  -35  
   2131 .. 2136  =  6 bp
promoter for the E. coli lac operon
lac promoter
2131 .. 2161  =  31 bp
   Segment 2:  
   2137 .. 2154  =  18 bp
promoter for the E. coli lac operon
lac promoter
2131 .. 2161  =  31 bp
   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
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