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Plasmid Files

pFA6a-kanMX6-PGAL1-3HA

Plasmid with a kanMX marker for swapping in the GAL1 promoter and adding a triple-HA tag.

 
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 BsiWI (25) PvuII (15) EcoO109I (4520) XmnI (4143) BpmI (3614) BmrI (3584) BanI (3492) AlwNI (3067) PspFI (2959) BseYI (2955) PciI (2651) BspQI - SapI (2535) HpaI (2472) SacII (2420) SfiI (2413) SpeI (2400) SalI (37) BstZ17I (177) PsiI (194) AscI - BssHII (251) BbvCI (257) R3 (261 .. 280) BamHI (297) PacI (371) BstAPI (522) BtgZI (541) AgeI (767) BglII (930) BstEII (960) BstXI (977) BmgBI (1013) MluI (1177) NcoI - StyI (1317) NruI (1401) EcoNI (1656) AsiSI (1744) PflMI (2007) PmeI (2365) Eco53kI (2372) SacI (2374) EcoRI (2376) F4 (2362 .. 2381) EcoRV (2390) pFA6a-kanMX6-PGAL1-3HA 4798 bp
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.
PvuII  (15)
1 site
C A G C T G G T C G A C
EcoO109I  (4520)
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.
XmnI  (4143)
1 site
G A A N N N N T T C C T T N N N N A A G
BpmI  (3614)
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  (3584)
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  (3492)
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  (3067)
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  (2959)
1 site
C C C A G C G G G T C G
BseYI  (2955)
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.
PciI  (2651)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2535)
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  (2535)
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.
HpaI  (2472)
1 site
G T T A A C C A A T T G
SacII  (2420)
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.
SfiI  (2413)
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.
SpeI  (2400)
1 site
A C T A G T T G A T C A
SalI  (37)
1 site
G T C G A C C A G C T G
BstZ17I  (177)
1 site
G T A T A C C A T A T G
PsiI  (194)
1 site
T T A T A A A A T A T T
AscI  (251)
1 site
G G C G C G C C C C G C G C G G
BssHII  (251)
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.
BbvCI  (257)
1 site
C C T C A G C G G A G T C G
BamHI  (297)
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.
PacI  (371)
1 site
T T A A T T A A A A T T A A T T
BstAPI  (522)
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.
BtgZI  (541)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its
electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
AgeI  (767)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BglII  (930)
1 site
A G A T C T T C T A G A
BstEII  (960)
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  (977)
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  (1013)
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.
MluI  (1177)
1 site
A C G C G T T G C G C A
NcoI  (1317)
1 site
C C A T G G G G T A C C
StyI  (1317)
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  (1401)
1 site
T C G C G A A G C G C T
EcoNI  (1656)
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  (1744)
1 site
G C G A T C G C C G C T A G C G
PflMI  (2007)
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  (2365)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (2372)
1 site
G A G C T C C T C G A G
SacI  (2374)
1 site
G A G C T C C T C G A G
EcoRI  (2376)
1 site
G A A T T C C T T A A G
EcoRV  (2390)
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.
R3
20-mer  /  55% GC
1 binding site
261 .. 280  =  20 annealed bases
Tm  =  58°C
Reverse primer for promoter swapping and adding
an N-terminal 3xHA tag. A gene-specific sequence
should be added at the 5' end of the primer.
F4
20-mer  /  40% GC
1 binding site
2362 .. 2381  =  20 annealed bases
Tm  =  52°C
Forward primer for promoter swapping. This primer
includes an EcoRI recognition sequence. A
gene-specific sequence should be added at the 5'
end of the primer.
kanMX
975 .. 2331  =  1357 bp
yeast selectable marker conferring kanamycin
resistance
kanMX
975 .. 2331  =  1357 bp
yeast selectable marker conferring kanamycin
resistance
AmpR
3471 .. 4331  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3471 .. 4262  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3471 .. 4331  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4263 .. 4331  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3471 .. 4331  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2712 .. 3300  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
2712 .. 3300  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
GAL1 promoter
400 .. 841  =  442 bp
inducible promoter, regulated by Gal4
GAL1 promoter
400 .. 841  =  442 bp
inducible promoter, regulated by Gal4
ADH1 terminator
50 .. 237  =  188 bp
ADH1 terminator
50 .. 237  =  188 bp
AmpR promoter
4332 .. 4436  =  105 bp
AmpR promoter
4332 .. 4436  =  105 bp
3xHA
270 .. 359  =  90 bp
30 amino acids  =  3.5 kDa
Product: three tandem HA epitope tags
3xHA
270 .. 359  =  90 bp
30 amino acids  =  3.5 kDa
Product: three tandem HA epitope tags
T7 promoter
2436 .. 2454  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
2436 .. 2454  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
4782 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4782 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
start codon
378 .. 380  =  3 bp
1 amino acid  =  149.2 Da
start codon
378 .. 380  =  3 bp
1 amino acid  =  149.2 Da
KanR
1319 .. 2128  =  810 bp
269 amino acids  =  30.7 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
1319 .. 2128  =  810 bp
269 amino acids  =  30.7 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
TEF promoter
975 .. 1318  =  344 bp
Ashbya gossypii TEF promoter
TEF promoter
975 .. 1318  =  344 bp
Ashbya gossypii TEF promoter
TEF terminator
2134 .. 2331  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
2134 .. 2331  =  198 bp
Ashbya gossypii TEF terminator
UAS
724 .. 841  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
UAS
724 .. 841  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
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