pFA6a-His3MX6-PGAL1-3HA

Plasmid with a HIS3MX6 marker for swapping in the GAL1 promoter and adding a triple-HA tag.
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HindIII (19) EcoO109I (4364) SspI (4192) XmnI (3987) ScaI (3868) PvuI (3758) BmrI (3428) BanI (3336) AlwNI (2911) PspFI (2803) BseYI (2799) BspQI - SapI (2379) HpaI (2316) SacII (2264) BsiWI (25) SalI (37) BstZ17I (177) AscI (251) BbvCI (257) R3 (261 .. 280) BamHI (297) PacI (371) BstAPI (522) AgeI (767) BglII (930) BstEII (960) BmgBI (1013) MluI (1177) NcoI - StyI (1317) SphI (1445) BclI * (1576) BsaBI (1578) NgoMIV (1646) NaeI (1648) XbaI (1697) AfeI (1885) PmeI (2209) Eco53kI (2216) SacI (2218) EcoRI (2220) F4 (2206 .. 2225) EcoRV (2234) SfiI (2257) pFA6a-His3MX6-PGAL1-3HA 4642 bp
HindIII  (19)
1 site
A A G C T T T T C G A A
EcoO109I  (4364)
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.
SspI  (4192)
1 site
A A T A T T T T A T A A
XmnI  (3987)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (3868)
1 site
A G T A C T T C A T G A
PvuI  (3758)
1 site
C G A T C G G C T A G C
BmrI  (3428)
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  (3336)
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  (2911)
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  (2803)
1 site
C C C A G C G G G T C G
BseYI  (2799)
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.
BspQI  (2379)
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  (2379)
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  (2316)
1 site
G T T A A C C A A T T G
SacII  (2264)
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.
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
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.
AgeI  (767)
1 site
A C C G G T T G G C C A
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.
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.
SphI  (1445)
1 site
G C A T G C C G T A C G
BclI  (1576)
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  (1578)
1 site
G A T N N N N A T C C T A N N N N T A G
NgoMIV  (1646)
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  (1648)
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  (1697)
1 site
T C T A G A A G A T C T
AfeI  (1885)
1 site
A G C G C T T C G C G A
PmeI  (2209)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (2216)
1 site
G A G C T C C T C G A G
SacI  (2218)
1 site
G A G C T C C T C G A G
EcoRI  (2220)
1 site
G A A T T C C T T A A G
EcoRV  (2234)
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.
SfiI  (2257)
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.
R3
20-mer  /  55% GC
1 binding site
261 .. 280  =  20 annealed bases
Tm  =  57°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
2206 .. 2225  =  20 annealed bases
Tm  =  53°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.
HIS3MX6
975 .. 2175  =  1201 bp
yeast selectable marker encoding the S. pombe his5 gene, which corresponds to S. cerevisiae HIS3
HIS3MX6
975 .. 2175  =  1201 bp
yeast selectable marker encoding the S. pombe his5 gene, which corresponds to S. cerevisiae HIS3
AmpR
3315 .. 4175  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3315 .. 4106  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3315 .. 4175  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   4107 .. 4175  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3315 .. 4175  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2556 .. 3144  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
2556 .. 3144  =  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
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
start codon
378 .. 380  =  3 bp
1 amino acid  =  149.2 Da
start codon
378 .. 380  =  3 bp
1 amino acid  =  149.2 Da
AmpR promoter
4176 .. 4280  =  105 bp
AmpR promoter
4176 .. 4280  =  105 bp
T7 promoter
2280 .. 2298  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
2280 .. 2298  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
4626 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4626 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
S. pombe his5
1319 .. 1972  =  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
1319 .. 1972  =  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
975 .. 1317  =  343 bp
Ashbya gossypii TEF promoter
TEF promoter
975 .. 1317  =  343 bp
Ashbya gossypii TEF promoter
TEF terminator
1978 .. 2175  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
1978 .. 2175  =  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
ORF:  916 .. 1185  =  270 bp
ORF:  89 amino acids  =  10.2 kDa
ORF:  3445 .. 3711  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1319 .. 1972  =  654 bp
ORF:  217 amino acids  =  23.6 kDa
ORF:  3315 .. 4175  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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