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Plasmid Files

pFA6a-His3MX6-PGAL1-GFP

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

 
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 HindIII (19) PfoI (5027) EcoO109I (4970) AatII (4916) ZraI (4914) SspI (4798) XmnI (4593) ScaI (4474) PvuI (4364) BmrI (4034) BanI (3942) BspQI - SapI (2985) SacII (2870) SfiI (2863) EcoRV (2840) F4 (2812 .. 2831) EcoRI (2826) SacI (2824) Eco53kI (2822) PmeI (2815) BsiWI (25) SalI (37) AscI (251) R5 (261 .. 280) BstBI (350) MfeI (410) PmlI (649) BsrGI (694) SnaBI (780) MscI (803) PacI (977) BstAPI (1128) AgeI (1373) BglII (1536) BstEII (1566) BmgBI (1619) Bpu10I (1628) MluI (1783) SphI (2051) BclI * (2182) BsaBI (2184) NgoMIV (2252) NaeI (2254) XbaI (2303) AfeI (2491) pFA6a-His3MX6-PGAL1-GFP 5248 bp
HindIII  (19)
1 site
A A G C T T T T C G A A
PfoI  (5027)
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  (4970)
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  (4916)
1 site
G A C G T C C T G C A G
ZraI  (4914)
1 site
G A C G T C C T G C A G
SspI  (4798)
1 site
A A T A T T T T A T A A
XmnI  (4593)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (4474)
1 site
A G T A C T T C A T G A
PvuI  (4364)
1 site
C G A T C G G C T A G C
BmrI  (4034)
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  (3942)
1 site
G G Y R C C C C R Y G G

Sticky ends from different BanI sites may not be compatible.
BspQI  (2985)
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  (2985)
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.
SacII  (2870)
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  (2863)
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  (2840)
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  (2826)
1 site
G A A T T C C T T A A G
SacI  (2824)
1 site
G A G C T C C T C G A G
Eco53kI  (2822)
1 site
G A G C T C C T C G A G
PmeI  (2815)
1 site
G T T T A A A C C A A A T T T 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
AscI  (251)
1 site
G G C G C G C C C C G C G C G G
BstBI  (350)
1 site
T T C G A A A A G C T T
MfeI  (410)
1 site
C A A T T G G T T A A C
PmlI  (649)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BsrGI  (694)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
SnaBI  (780)
1 site
T A C G T A A T G C A T
MscI  (803)
1 site
T G G C C A A C C G G T
PacI  (977)
1 site
T T A A T T A A A A T T A A T T
BstAPI  (1128)
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  (1373)
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  (1536)
1 site
A G A T C T T C T A G A
BstEII  (1566)
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  (1619)
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  (1628)
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  (1783)
1 site
A C G C G T T G C G C A
SphI  (2051)
1 site
G C A T G C C G T A C G
BclI  (2182)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
BsaBI  (2184)
1 site
G A T N N N N A T C C T A N N N N T A G
NgoMIV  (2252)
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  (2254)
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  (2303)
1 site
T C T A G A A G A T C T
AfeI  (2491)
1 site
A G C G C T T C G C G A
F4
20-mer  /  40% GC
1 binding site
2812 .. 2831  =  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.
R5
20-mer  /  35% GC
1 binding site
261 .. 280  =  20 annealed bases
Tm  =  49°C
Reverse primer for promoter swapping and adding
an N-terminal GFP tag. A gene-specific sequence
should be added at the 5' end of the primer.
HIS3MX6
1581 .. 2781  =  1201 bp
yeast selectable marker encoding the S. pombe his5
gene, which corresponds to S. cerevisiae HIS3
HIS3MX6
1581 .. 2781  =  1201 bp
yeast selectable marker encoding the S. pombe his5
gene, which corresponds to S. cerevisiae HIS3
AmpR
3921 .. 4781  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3921 .. 4712  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3921 .. 4781  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4713 .. 4781  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3921 .. 4781  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
GFP(S65T)
258 .. 970  =  713 bp
236 amino acids  =  26.7 kDa
Product: green fluorescent protein, S65T variant
GFP(S65T)
258 .. 970  =  713 bp
236 amino acids  =  26.7 kDa
Product: green fluorescent protein, S65T variant
ori
3162 .. 3750  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
3162 .. 3750  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
GAL1 promoter
1006 .. 1447  =  442 bp
inducible promoter, regulated by Gal4
GAL1 promoter
1006 .. 1447  =  442 bp
inducible promoter, regulated by Gal4
ADH1 terminator
50 .. 237  =  188 bp
ADH1 terminator
50 .. 237  =  188 bp
AmpR promoter
4782 .. 4886  =  105 bp
AmpR promoter
4782 .. 4886  =  105 bp
T7 promoter
2886 .. 2904  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
2886 .. 2904  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
5232 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
5232 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
start codon
984 .. 986  =  3 bp
1 amino acid  =  149.2 Da
start codon
984 .. 986  =  3 bp
1 amino acid  =  149.2 Da
S. pombe his5
1925 .. 2578  =  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
1925 .. 2578  =  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
1581 .. 1923  =  343 bp
Ashbya gossypii TEF promoter
TEF promoter
1581 .. 1923  =  343 bp
Ashbya gossypii TEF promoter
TEF terminator
2584 .. 2781  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
2584 .. 2781  =  198 bp
Ashbya gossypii TEF terminator
UAS
1330 .. 1447  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
UAS
1330 .. 1447  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
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