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Plasmid Files

pFA6a-His3MX6-PGAL1-GST

Plasmid with a HIS3MX6 marker for swapping in the GAL1 promoter and adding a GST tag.

 
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 HindIII (19) NdeI (5126) PfoI (4988) AatII (4877) ZraI (4875) SspI (4759) PvuI (4325) BmrI (3995) BanI (3903) AlwNI (3478) PspFI (3370) BseYI (3366) HpaI (2883) SacII (2831) SfiI (2824) EcoRV (2801) F4 (2773 .. 2792) EcoRI (2787) SacI (2785) Eco53kI (2783) BsiWI (25) SalI (37) BstZ17I (177) AscI (251) R4 (261 .. 280) SwaI (504) BstBI (532) BsgI (647) MscI (724) EcoNI (920) PacI (938) BstAPI (1089) AgeI (1334) BglII (1497) BstEII (1527) BmgBI (1580) Bpu10I (1589) MluI (1744) NcoI - StyI (1884) SphI (2012) BsaBI (2145) NgoMIV (2213) NaeI (2215) XbaI (2264) AfeI (2452) PmeI (2776) pFA6a-His3MX6-PGAL1-GST 5209 bp
HindIII  (19)
1 site
A A G C T T T T C G A A
NdeI  (5126)
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  (4988)
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.
AatII  (4877)
1 site
G A C G T C C T G C A G
ZraI  (4875)
1 site
G A C G T C C T G C A G
SspI  (4759)
1 site
A A T A T T T T A T A A
PvuI  (4325)
1 site
C G A T C G G C T A G C
BmrI  (3995)
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  (3903)
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  (3478)
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  (3370)
1 site
C C C A G C G G G T C G
BseYI  (3366)
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.
HpaI  (2883)
1 site
G T T A A C C A A T T G
SacII  (2831)
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  (2824)
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.
EcoRV  (2801)
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.
EcoRI  (2787)
1 site
G A A T T C C T T A A G
SacI  (2785)
1 site
G A G C T C C T C G A G
Eco53kI  (2783)
1 site
G A G C T C C T C G A G
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
BstZ17I  (177)
1 site
G T A T A C C A T A T G
AscI  (251)
1 site
G G C G C G C C C C G C G C G G
SwaI  (504)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
BstBI  (532)
1 site
T T C G A A A A G C T T
BsgI  (647)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
MscI  (724)
1 site
T G G C C A A C C G G T
EcoNI  (920)
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.
PacI  (938)
1 site
T T A A T T A A A A T T A A T T
BstAPI  (1089)
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.
AgeI  (1334)
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  (1497)
1 site
A G A T C T T C T A G A
BstEII  (1527)
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.
BmgBI  (1580)
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.
Bpu10I  (1589)
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.
MluI  (1744)
1 site
A C G C G T T G C G C A
NcoI  (1884)
1 site
C C A T G G G G T A C C
StyI  (1884)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
SphI  (2012)
1 site
G C A T G C C G T A C G
BsaBI  (2145)
1 site
G A T N N N N A T C C T A N N N N T A G
NgoMIV  (2213)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
NaeI  (2215)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
XbaI  (2264)
1 site
T C T A G A A G A T C T
AfeI  (2452)
1 site
A G C G C T T C G C G A
PmeI  (2776)
1 site
G T T T A A A C C A A A T T T G
F4
20-mer  /  40% GC
1 binding site
2773 .. 2792  =  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.
R4
20-mer  /  55% GC
1 binding site
261 .. 280  =  20 annealed bases
Tm  =  62°C
Reverse primer for promoter swapping and adding
an N-terminal GST tag. A gene-specific sequence
should be added at the 5' end of the primer.
HIS3MX6
1542 .. 2742  =  1201 bp
yeast selectable marker encoding the S. pombe his5
gene, which corresponds to S. cerevisiae HIS3
HIS3MX6
1542 .. 2742  =  1201 bp
yeast selectable marker encoding the S. pombe his5
gene, which corresponds to S. cerevisiae HIS3
AmpR
3882 .. 4742  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3882 .. 4673  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3882 .. 4742  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4674 .. 4742  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3882 .. 4742  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
GST
279 .. 932  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
GST
279 .. 932  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
ori
3123 .. 3711  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
3123 .. 3711  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
GAL1 promoter
967 .. 1408  =  442 bp
inducible promoter, regulated by Gal4
GAL1 promoter
967 .. 1408  =  442 bp
inducible promoter, regulated by Gal4
ADH1 terminator
50 .. 237  =  188 bp
ADH1 terminator
50 .. 237  =  188 bp
AmpR promoter
4743 .. 4847  =  105 bp
AmpR promoter
4743 .. 4847  =  105 bp
T7 promoter
2847 .. 2865  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
2847 .. 2865  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
5193 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
5193 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
start codon
945 .. 947  =  3 bp
1 amino acid  =  149.2 Da
start codon
945 .. 947  =  3 bp
1 amino acid  =  149.2 Da
S. pombe his5
1886 .. 2539  =  654 bp
217 amino acids  =  23.6 kDa
Product: imidazoleglycerol-phosphate dehydratase,
required for histidine biosynthesis
yeast auxotrophic marker; corresponds to S.
cerevisiae HIS3
S. pombe his5
1886 .. 2539  =  654 bp
217 amino acids  =  23.6 kDa
Product: imidazoleglycerol-phosphate dehydratase,
required for histidine biosynthesis
yeast auxotrophic marker; corresponds to S.
cerevisiae HIS3
TEF promoter
1542 .. 1884  =  343 bp
Ashbya gossypii TEF promoter
TEF promoter
1542 .. 1884  =  343 bp
Ashbya gossypii TEF promoter
TEF terminator
2545 .. 2742  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
2545 .. 2742  =  198 bp
Ashbya gossypii TEF terminator
UAS
1291 .. 1408  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
UAS
1291 .. 1408  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
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