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Plasmid Files

pFA6a-TRP1-PGAL1-GFP

Plasmid with a TRP1 marker for swapping in the GAL1 promoter and adding a GFP tag.

 
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pFA6a-TRP1-PGAL1-GFP.dna
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BsiWI (25) PfoI (4660) EcoO109I (4603) AatII (4549) ZraI (4547) SspI (4431) ScaI (4107) PvuI (3997) FspI (3849) BanI (3575) PciI (2734) BspQI - SapI (2618) SacII (2503) SfiI (2496) SpeI (2483) BspDI - ClaI (2466) F4 (2445 .. 2464) EcoRI (2459) SacI (2457) PstI (35) SalI (37) PsiI (194) AscI - BssHII (251) R5 (261 .. 280) BstBI (350) SnaBI (780) MscI (803) NcoI - StyI (804) PacI (977) AgeI (1373) BseRI (1411) BglII (1536) BspEI * (1554) XbaI (1773) Bsu36I (1931) BstXI (1987) BsgI (2102) AarI (2122) PmeI (2448) Eco53kI (2455) pFA6a-TRP1-PGAL1-GFP 4881 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.
PfoI  (4660)
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  (4603)
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  (4549)
1 site
G A C G T C C T G C A G
ZraI  (4547)
1 site
G A C G T C C T G C A G
SspI  (4431)
1 site
A A T A T T T T A T A A
ScaI  (4107)
1 site
A G T A C T T C A T G A
PvuI  (3997)
1 site
C G A T C G G C T A G C
FspI  (3849)
1 site
T G C G C A A C G C G T
BanI  (3575)
1 site
G G Y R C C C C R Y G G

Sticky ends from different BanI sites may not be compatible.
PciI  (2734)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2618)
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  (2618)
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  (2503)
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  (2496)
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  (2483)
1 site
A C T A G T T G A T C A
BspDI  (2466)
1 site
A T C G A T T A G C T A
ClaI  (2466)
1 site
A T C G A T T A G C T A
EcoRI  (2459)
1 site
G A A T T C C T T A A G
SacI  (2457)
1 site
G A G C T C C T C G A G
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.
BstBI  (350)
1 site
T T C G A A A A G C T T
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
NcoI  (804)
1 site
C C A T G G G G T A C C
StyI  (804)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
PacI  (977)
1 site
T T A A T T A A A A T T A A T T
AgeI  (1373)
1 site
A C C G G T T G G C C A
BseRI  (1411)
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  (1536)
1 site
A G A T C T T C T A G A
BspEI  (1554)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
XbaI  (1773)
1 site
T C T A G A A G A T C T
Bsu36I  (1931)
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  (1987)
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  (2102)
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  (2122)
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  (2448)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (2455)
1 site
G A G C T C C T C G A G
F4
20-mer  /  40% GC
1 binding site
2445 .. 2464  =  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.
AmpR
3554 .. 4414  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3554 .. 4345  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3554 .. 4414  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4346 .. 4414  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3554 .. 4414  =  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
TRP1
1690 .. 2364  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis
yeast auxotrophic marker
TRP1
1690 .. 2364  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis
yeast auxotrophic marker
ori
2795 .. 3383  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
2795 .. 3383  =  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
TRP1 promoter
1542 .. 1689  =  148 bp
TRP1 promoter
1542 .. 1689  =  148 bp
AmpR promoter
4415 .. 4519  =  105 bp
AmpR promoter
4415 .. 4519  =  105 bp
T7 promoter
2519 .. 2537  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
2519 .. 2537  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
4865 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4865 .. 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
UAS
1330 .. 1447  =  118 bp
upstream activating sequence mediating Gal4-dependent induction
UAS
1330 .. 1447  =  118 bp
upstream activating sequence mediating Gal4-dependent induction
ORF:  1690 .. 2364  =  675 bp
ORF:  224 amino acids  =  24.1 kDa
ORF:  1940 .. 2221  =  282 bp
ORF:  93 amino acids  =  10.8 kDa
ORF:  3684 .. 3950  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  258 .. 986  =  729 bp
ORF:  242 amino acids  =  27.4 kDa
ORF:  3554 .. 4414  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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