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Plasmid Files

pAG29

Plasmid for yeast gene deletion using the patMX4 selectable marker conferring bialaphos resistance.

 
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pAG29.dna
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PvuII (15) NdeI (3601) EcoO109I (3406) AatII (3352) ZraI (3350) SspI (3234) XmnI (3029) ScaI (2910) TsoI (2829) PvuI (2800) AlwNI (1953) PspFI (1845) HindIII (19) BamHI (43) TspMI - XmaI (48) SmaI (50) PacI (58) AscI (63) BstEII (100) BstXI (117) Bpu10I (162) BseRI (264) MluI (317) PflFI - Tth111I (529) BtgZI (654) AarI (794) NcoI - StyI (797) PasI (807) BsgI (850) EcoNI (868) MauBI (889) DraIII (939) BsmI (1201) Eco53kI (1258) SacI (1260) ApoI - EcoRI (1262) BspDI - ClaI (1269) EcoRV (1276) SpeI (1286) SfiI (1299) SacII (1306) HpaI (1358) BspQI - SapI (1421) PciI (1537) BseYI (1841) pAG29 3684 bp
PvuII  (15)
1 site
C A G C T G G T C G A C
NdeI  (3601)
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.
EcoO109I  (3406)
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  (3352)
1 site
G A C G T C C T G C A G
ZraI  (3350)
1 site
G A C G T C C T G C A G
SspI  (3234)
1 site
A A T A T T T T A T A A
XmnI  (3029)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (2910)
1 site
A G T A C T T C A T G A
TsoI  (2829)
1 site
T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9

Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
PvuI  (2800)
1 site
C G A T C G G C T A G C
AlwNI  (1953)
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  (1845)
1 site
C C C A G C G G G T C G
HindIII  (19)
1 site
A A G C T T T T C G A A
BamHI  (43)
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.
TspMI  (48)
1 site
C C C G G G G G G C C C
XmaI  (48)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (50)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
PacI  (58)
1 site
T T A A T T A A A A T T A A T T
AscI  (63)
1 site
G G C G C G C C C C G C G C G G
BstEII  (100)
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  (117)
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.
Bpu10I  (162)
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.
BseRI  (264)
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.
MluI  (317)
1 site
A C G C G T T G C G C A
PflFI  (529)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (529)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
BtgZI  (654)
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.
AarI  (794)
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.
NcoI  (797)
1 site
C C A T G G G G T A C C
StyI  (797)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
PasI  (807)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
BsgI  (850)
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).
EcoNI  (868)
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.
MauBI  (889)
1 site
C G C G C G C G G C G C G C G C
DraIII  (939)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
BsmI  (1201)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
Eco53kI  (1258)
1 site
G A G C T C C T C G A G
SacI  (1260)
1 site
G A G C T C C T C G A G
ApoI  (1262)
1 site
R A A T T Y Y T T A A R

ApoI is typically used at 50°C, but is 50% active at 37°C.
EcoRI  (1262)
1 site
G A A T T C C T T A A G
BspDI  (1269)
1 site
A T C G A T T A G C T A
ClaI  (1269)
1 site
A T C G A T T A G C T A
EcoRV  (1276)
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.
SpeI  (1286)
1 site
A C T A G T T G A T C A
SfiI  (1299)
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.
SacII  (1306)
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  (1358)
1 site
G T T A A C C A A T T G
BspQI  (1421)
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  (1421)
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.
PciI  (1537)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BseYI  (1841)
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.
patMX4
115 .. 1214  =  1100 bp
Product: yeast selectable marker conferring bialaphos resistance (Goldstein and McCusker, 1999)
patMX4
115 .. 1214  =  1100 bp
Product: yeast selectable marker conferring bialaphos resistance (Goldstein and McCusker, 1999)
AmpR
2357 .. 3217  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2357 .. 3148  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2357 .. 3217  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3149 .. 3217  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2357 .. 3217  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1598 .. 2186  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1598 .. 2186  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
3218 .. 3322  =  105 bp
AmpR promoter
3218 .. 3322  =  105 bp
T7 promoter
1322 .. 1340  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1322 .. 1340  =  19 bp
promoter for bacteriophage T7 RNA polymerase
BlpR
511 .. 966  =  456 bp
151 amino acids  =  17.1 kDa
Product: phosphinothricin acetyltransferase
confers resistance to bialaphos or phosphinothricin
BlpR
511 .. 966  =  456 bp
151 amino acids  =  17.1 kDa
Product: phosphinothricin acetyltransferase
confers resistance to bialaphos or phosphinothricin
TEF promoter
115 .. 458  =  344 bp
Ashbya gossypii TEF promoter
TEF promoter
115 .. 458  =  344 bp
Ashbya gossypii TEF promoter
TEF terminator
1017 .. 1214  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
1017 .. 1214  =  198 bp
Ashbya gossypii TEF terminator
ORF:  511 .. 966  =  456 bp
ORF:  151 amino acids  =  17.1 kDa
ORF:  2487 .. 2753  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3680 .. 229  =  234 bp
ORF:  77 amino acids  =  8.3 kDa
ORF:  2357 .. 3217  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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