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Plasmid Files

pFA6a-kanMX6

Plasmid for yeast gene deletion using the kanMX selectable marker conferring kanamycin resistance.

 
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pFA6a-kanMX6.dna
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PvuII (15) NdeI (3855) PfoI (3717) EcoO109I (3660) AatII (3606) ZraI (3604) XmnI (3283) NmeAIII (2832) BpmI (2754) BmrI (2724) BanI (2632) AlwNI (2207) PspFI (2099) BseYI (2095) BsiWI (25) SalI (37) AccI (38) F1 (42 .. 61) BamHI (43) AvaI - BsoBI - TspMI - XmaI (48) BmeT110I (49) SmaI (50) PacI (58) AscI - BssHII (63) BglII (70) BstEII (100) BstXI (117) BmgBI (153) BseRI (264) MluI (317) NcoI - StyI (457) NruI (541) EcoNI (796) AsiSI (884) PflMI (1147) PmeI (1505) Eco53kI (1512) SacI (1514) EcoRI (1516) R1 (1502 .. 1521) EcoRV (1530) SpeI (1540) SfiI (1553) SacII (1560) HpaI (1612) BspQI - SapI (1675) PciI (1791) pFA6a-kanMX6 3938 bp
PvuII  (15)
1 site
C A G C T G G T C G A C
NdeI  (3855)
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.
PfoI  (3717)
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.
EcoO109I  (3660)
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.
AatII  (3606)
1 site
G A C G T C C T G C A G
ZraI  (3604)
1 site
G A C G T C C T G C A G
XmnI  (3283)
1 site
G A A N N N N T T C C T T N N N N A A G
NmeAIII  (2832)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BpmI  (2754)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
BmrI  (2724)
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.
BanI  (2632)
1 site
G G Y R C C C C R Y G G

Sticky ends from different BanI sites may not be compatible.
AlwNI  (2207)
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.
PspFI  (2099)
1 site
C C C A G C G G G T C G
BseYI  (2095)
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.
BsiWI  (25)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
SalI  (37)
1 site
G T C G A C C A G C T G
AccI  (38)
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.
BamHI  (43)
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  (48)
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  (48)
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  (48)
1 site
C C C G G G G G G C C C
XmaI  (48)
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  (49)
1 site
C Y C G R G G R G C Y C
SmaI  (50)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
PacI  (58)
1 site
T T A A T T A A A A T T A A T T
AscI  (63)
1 site
G G C G C G C C C C G C G C G G
BssHII  (63)
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.
BglII  (70)
1 site
A G A T C T T C T A G A
BstEII  (100)
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.
BstXI  (117)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
BmgBI  (153)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
BseRI  (264)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
MluI  (317)
1 site
A C G C G T T G C G C A
NcoI  (457)
1 site
C C A T G G G G T A C C
StyI  (457)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
NruI  (541)
1 site
T C G C G A A G C G C T
EcoNI  (796)
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.
AsiSI  (884)
1 site
G C G A T C G C C G C T A G C G
PflMI  (1147)
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.
PmeI  (1505)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (1512)
1 site
G A G C T C C T C G A G
SacI  (1514)
1 site
G A G C T C C T C G A G
EcoRI  (1516)
1 site
G A A T T C C T T A A G
EcoRV  (1530)
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.
SpeI  (1540)
1 site
A C T A G T T G A T C A
SfiI  (1553)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
SacII  (1560)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
HpaI  (1612)
1 site
G T T A A C C A A T T G
BspQI  (1675)
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  (1675)
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.
PciI  (1791)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
F1
20-mer  /  50% GC
1 binding site
42 .. 61  =  20 annealed bases
Tm  =  54°C
Forward primer for gene deletion. This primer includes a BamHI recognition sequence. A gene-specific sequence should be added at the 5' end of the primer.
R1
20-mer  /  40% GC
1 binding site
1502 .. 1521  =  20 annealed bases
Tm  =  52°C
Reverse primer for gene deletion or C-terminal tagging. This primer includes an EcoRI recognition sequence. A gene-specific sequence should be added at the 5' end of the primer.
kanMX
115 .. 1471  =  1357 bp
yeast selectable marker conferring kanamycin resistance
kanMX
115 .. 1471  =  1357 bp
yeast selectable marker conferring kanamycin resistance
AmpR
2611 .. 3471  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2611 .. 3402  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2611 .. 3471  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3403 .. 3471  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2611 .. 3471  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1852 .. 2440  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
1852 .. 2440  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
3472 .. 3576  =  105 bp
AmpR promoter
3472 .. 3576  =  105 bp
T7 promoter
1576 .. 1594  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1576 .. 1594  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
3922 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
3922 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
KanR
459 .. 1268  =  810 bp
269 amino acids  =  30.7 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
459 .. 1268  =  810 bp
269 amino acids  =  30.7 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
TEF promoter
115 .. 458  =  344 bp
Ashbya gossypii TEF promoter
TEF promoter
115 .. 458  =  344 bp
Ashbya gossypii TEF promoter
TEF terminator
1274 .. 1471  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
1274 .. 1471  =  198 bp
Ashbya gossypii TEF terminator
ORF:  2741 .. 3007  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  459 .. 1268  =  810 bp
ORF:  269 amino acids  =  30.7 kDa
ORF:  3934 .. 229  =  234 bp
ORF:  77 amino acids  =  8.3 kDa
ORF:  2611 .. 3471  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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