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Plasmid Files

pFA6a-TRP1-PGAL1-GST

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

 
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pFA6a-TRP1-PGAL1-GST.dna
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PvuII (15) NdeI (4759) PfoI (4621) AatII (4510) ZraI (4508) SspI (4392) PvuI (3958) FspI (3810) BpmI (3658) BanI (3536) AlwNI (3111) HpaI (2516) SacII (2464) BtgI (2461) SfiI (2457) SpeI (2444) BspDI - ClaI (2427) F4 (2406 .. 2425) BsiWI (25) PstI (35) SalI (37) PsiI (194) AscI - BssHII (251) R4 (261 .. 280) BclI * (493) SwaI (504) BstBI (532) MscI (724) EcoNI (920) PacI (938) AgeI (1334) BseRI (1372) BglII (1497) BsrGI (1502) BspEI * (1515) PmlI (1610) XbaI (1734) MfeI (1790) Bsu36I (1892) BstXI (1948) AarI (2083) PmeI (2409) Eco53kI (2416) SacI (2418) EcoRI (2420) pFA6a-TRP1-PGAL1-GST 4842 bp
PvuII  (15)
1 site
C A G C T G G T C G A C
NdeI  (4759)
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  (4621)
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  (4510)
1 site
G A C G T C C T G C A G
ZraI  (4508)
1 site
G A C G T C C T G C A G
SspI  (4392)
1 site
A A T A T T T T A T A A
PvuI  (3958)
1 site
C G A T C G G C T A G C
FspI  (3810)
1 site
T G C G C A A C G C G T
BpmI  (3658)
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  (3536)
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  (3111)
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.
HpaI  (2516)
1 site
G T T A A C C A A T T G
SacII  (2464)
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.
BtgI  (2461)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
SfiI  (2457)
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  (2444)
1 site
A C T A G T T G A T C A
BspDI  (2427)
1 site
A T C G A T T A G C T A
ClaI  (2427)
1 site
A T C G A T T A G C T A
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.
BclI  (493)
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.
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
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
AgeI  (1334)
1 site
A C C G G T T G G C C A
BseRI  (1372)
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  (1497)
1 site
A G A T C T T C T A G A
BsrGI  (1502)
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  (1515)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
PmlI  (1610)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
XbaI  (1734)
1 site
T C T A G A A G A T C T
MfeI  (1790)
1 site
C A A T T G G T T A A C
Bsu36I  (1892)
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  (1948)
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.
AarI  (2083)
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  (2409)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (2416)
1 site
G A G C T C C T C G A G
SacI  (2418)
1 site
G A G C T C C T C G A G
EcoRI  (2420)
1 site
G A A T T C C T T A A G
F4
20-mer  /  40% GC
1 binding site
2406 .. 2425  =  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.
AmpR
3515 .. 4375  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3515 .. 4306  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3515 .. 4375  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4307 .. 4375  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3515 .. 4375  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
TRP1
1651 .. 2325  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis
yeast auxotrophic marker
TRP1
1651 .. 2325  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis
yeast auxotrophic marker
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
2756 .. 3344  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
2756 .. 3344  =  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
TRP1 promoter
1503 .. 1650  =  148 bp
TRP1 promoter
1503 .. 1650  =  148 bp
AmpR promoter
4376 .. 4480  =  105 bp
AmpR promoter
4376 .. 4480  =  105 bp
T7 promoter
2480 .. 2498  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
2480 .. 2498  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
4826 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4826 .. 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
UAS
1291 .. 1408  =  118 bp
upstream activating sequence mediating Gal4-dependent induction
UAS
1291 .. 1408  =  118 bp
upstream activating sequence mediating Gal4-dependent induction
ORF:  1651 .. 2325  =  675 bp
ORF:  224 amino acids  =  24.1 kDa
ORF:  1901 .. 2182  =  282 bp
ORF:  93 amino acids  =  10.8 kDa
ORF:  3645 .. 3911  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  258 .. 947  =  690 bp
ORF:  229 amino acids  =  26.7 kDa
ORF:  3515 .. 4375  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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