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Plasmid Files

pFA6a-kanMX6-PGAL1-GFP

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

 
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pFA6a-kanMX6-PGAL1-GFP.dna
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BsiWI (25) PfoI (5183) EcoO109I (5126) AatII (5072) ZraI (5070) XmnI (4749) BmrI (4190) BanI (4098) PciI (3257) BspQI - SapI (3141) SacII (3026) SfiI (3019) SpeI (3006) EcoRV (2996) F4 (2968 .. 2987) EcoRI (2982) SacI (2980) Eco53kI (2978) PmeI (2971) SalI (37) PsiI (194) AscI - BssHII (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) BstXI (1583) BmgBI (1619) MluI (1783) NruI (2007) EcoNI (2262) AsiSI (2350) PflMI (2613) pFA6a-kanMX6-PGAL1-GFP 5404 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  (5183)
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  (5126)
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  (5072)
1 site
G A C G T C C T G C A G
ZraI  (5070)
1 site
G A C G T C C T G C A G
XmnI  (4749)
1 site
G A A N N N N T T C C T T N N N N A A G
BmrI  (4190)
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  (4098)
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  (3257)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3141)
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  (3141)
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  (3026)
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  (3019)
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  (3006)
1 site
A C T A G T T G A T C A
EcoRV  (2996)
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  (2982)
1 site
G A A T T C C T T A A G
SacI  (2980)
1 site
G A G C T C C T C G A G
Eco53kI  (2978)
1 site
G A G C T C C T C G A G
PmeI  (2971)
1 site
G T T T A A A C C A A A T T T G
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
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
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.
BstXI  (1583)
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.
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.
MluI  (1783)
1 site
A C G C G T T G C G C A
NruI  (2007)
1 site
T C G C G A A G C G C T
EcoNI  (2262)
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.
AsiSI  (2350)
1 site
G C G A T C G C C G C T A G C G
PflMI  (2613)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
F4
20-mer  /  40% GC
1 binding site
2968 .. 2987  =  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.
kanMX
1581 .. 2937  =  1357 bp
yeast selectable marker conferring kanamycin resistance
kanMX
1581 .. 2937  =  1357 bp
yeast selectable marker conferring kanamycin resistance
AmpR
4077 .. 4937  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4077 .. 4868  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4077 .. 4937  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4869 .. 4937  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4077 .. 4937  =  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
3318 .. 3906  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
3318 .. 3906  =  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
4938 .. 5042  =  105 bp
AmpR promoter
4938 .. 5042  =  105 bp
T7 promoter
3042 .. 3060  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3042 .. 3060  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
5388 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
5388 .. 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
KanR
1925 .. 2734  =  810 bp
269 amino acids  =  30.7 kDa
Product: aminoglycoside phosphotransferase
confers