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Plasmid Files

pFA6a-TRP1-PGAL1-3HA

Plasmid with a TRP1 marker for swapping in the GAL1 promoter and adding a triple-HA tag.

 
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 PvuII (15) EcoO109I (3997) SspI (3825) ScaI (3501) PvuI (3391) FspI (3243) BpmI (3091) BanI (2969) AlwNI (2544) AflIII - PciI (2128) BsiWI (25) PstI (35) SalI (37) PsiI (194) AscI - BssHII (251) BbvCI - Bpu10I (257) R3 (261 .. 280) BamHI (297) PacI (371) BtgZI (541) AgeI (767) BseRI (805) BglII (930) BsrGI (935) BspEI * (948) PmlI (1043) XbaI (1167) MfeI (1223) Bsu36I (1325) BstXI (1381) BsgI (1496) AarI (1516) PmeI (1842) Eco53kI (1849) SacI (1851) EcoRI (1853) F4 (1839 .. 1858) BspDI - ClaI (1860) SpeI (1877) SfiI (1890) BtgI (1894) SacII (1897) HpaI (1949) BspQI - SapI (2012) pFA6a-TRP1-PGAL1-3HA 4275 bp
PvuII  (15)
1 site
C A G C T G G T C G A C
EcoO109I  (3997)
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  (3825)
1 site
A A T A T T T T A T A A
ScaI  (3501)
1 site
A G T A C T T C A T G A
PvuI  (3391)
1 site
C G A T C G G C T A G C
FspI  (3243)
1 site
T G C G C A A C G C G T
BpmI  (3091)
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.
BanI  (2969)
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  (2544)
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.
AflIII  (2128)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (2128)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
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.
PstI  (35)
1 site
C T G C A G G A C G T C
SalI  (37)
1 site
G T C G A C C A G C 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
Bpu10I  (257)
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.
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
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.
BseRI  (805)
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.
BglII  (930)
1 site
A G A T C T T C T A G A
BsrGI  (935)
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.
BspEI  (948)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
PmlI  (1043)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
XbaI  (1167)
1 site
T C T A G A A G A T C T
MfeI  (1223)
1 site
C A A T T G G T T A A C
Bsu36I  (1325)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BstXI  (1381)
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.
BsgI  (1496)
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).
AarI  (1516)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
PmeI  (1842)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (1849)
1 site
G A G C T C C T C G A G
SacI  (1851)
1 site
G A G C T C C T C G A G
EcoRI  (1853)
1 site
G A A T T C C T T A A G
BspDI  (1860)
1 site
A T C G A T T A G C T A
ClaI  (1860)
1 site
A T C G A T T A G C T A
SpeI  (1877)
1 site
A C T A G T T G A T C A
SfiI  (1890)
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.
BtgI  (1894)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
SacII  (1897)
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  (1949)
1 site
G T T A A C C A A T T G
BspQI  (2012)
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  (2012)
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.
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
1839 .. 1858  =  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.
AmpR
2948 .. 3808  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2948 .. 3739  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2948 .. 3808  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3740 .. 3808  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2948 .. 3808  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TRP1
1084 .. 1758  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase,
required for tryptophan biosynthesis
yeast auxotrophic marker
TRP1
1084 .. 1758  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase,
required for tryptophan biosynthesis
yeast auxotrophic marker
ori
2189 .. 2777  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
2189 .. 2777  =  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
TRP1 promoter
936 .. 1083  =  148 bp
TRP1 promoter
936 .. 1083  =  148 bp
AmpR promoter
3809 .. 3913  =  105 bp
AmpR promoter
3809 .. 3913  =  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
1913 .. 1931  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1913 .. 1931  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
4259 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4259 .. 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
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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