pUCATPH

Hygromycin resistance plasmid for restriction enzyme-mediated integration (REMI) mutagenesis of fungi.
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SspI (4903) AhdI (4099) AlwNI (3622) PciI (3206) BspQI - SapI (3090) SacI (2848) Eco53kI (2846) KpnI (2842) Acc65I (2838) SmaI (2836) AvaI - BsoBI - TspMI - XmaI (2834) PfoI (46) HindIII (399) SbfI (415) EcoNI (445) AfeI (576) SgrAI (658) BstZ17I (836) PasI (939) BseRI (1002) BlpI (1121) MluI (1225) SacII (1677) BtgZI (1779) RsrII (2044) AsiSI (2093) PshAI (2428) BspDI - ClaI (2458) AflII (2519) pUCATPH 5086 bp
SspI  (4903)
1 site
A A T A T T T T A T A A
AhdI  (4099)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
AlwNI  (3622)
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.
PciI  (3206)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3090)
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  (3090)
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.
SacI  (2848)
1 site
G A G C T C C T C G A G
Eco53kI  (2846)
1 site
G A G C T C C T C G A G
KpnI  (2842)
1 site
G G T A C C C C A T G G
Acc65I  (2838)
1 site
G G T A C C C C A T G G
SmaI  (2836)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
AvaI  (2834)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (2834)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
TspMI  (2834)
1 site
C C C G G G G G G C C C
XmaI  (2834)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
PfoI  (46)
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.
HindIII  (399)
1 site
A A G C T T T T C G A A
SbfI  (415)
1 site
C C T G C A G G G G A C G T C C
EcoNI  (445)
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.
AfeI  (576)
1 site
A G C G C T T C G C G A
SgrAI  (658)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
BstZ17I  (836)
1 site
G T A T A C C A T A T G
PasI  (939)
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.
BseRI  (1002)
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.
BlpI  (1121)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
MluI  (1225)
1 site
A C G C G T T G C G C A
SacII  (1677)
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.
BtgZI  (1779)
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.
RsrII  (2044)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
AsiSI  (2093)
1 site
G C G A T C G C C G C T A G C G
PshAI  (2428)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
BspDI  (2458)
1 site
A T C G A T T A G C T A
ClaI  (2458)
1 site
A T C G A T T A G C T A
AflII  (2519)
1 site
C T T A A G G A A T T C
HygR
1431 .. 2456  =  1026 bp
341 amino acids  =  38.0 kDa
Product: aminoglycoside phosphotransferase from E. coli
confers resistance to hygromycin
HygR
1431 .. 2456  =  1026 bp
341 amino acids  =  38.0 kDa
Product: aminoglycoside phosphotransferase from E. coli
confers resistance to hygromycin
AmpR
4026 .. 4886  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   4026 .. 4817  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4026 .. 4886  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   4818 .. 4886  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4026 .. 4886  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3267 .. 3855  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3267 .. 3855  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
trpC terminator
704 .. 1262  =  559 bp
terminator for Aspergillus nidulans trpC
trpC terminator
704 .. 1262  =  559 bp
terminator for Aspergillus nidulans trpC
trpC promoter
2461 .. 2817  =  357 bp
promoter for Aspergillus nidulans trpC
trpC promoter
2461 .. 2817  =  357 bp
promoter for Aspergillus nidulans trpC
AmpR promoter
4887 .. 4991  =  105 bp
AmpR promoter
4887 .. 4991  =  105 bp
ORF:  508 .. 771  =  264 bp
ORF:  87 amino acids  =  9.0 kDa
ORF:  1390 .. 1632  =  243 bp
ORF:  80 amino acids  =  8.4 kDa
ORF:  4156 .. 4422  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  407 .. 634  =  228 bp
ORF:  75 amino acids  =  7.9 kDa
ORF:  186 .. 473  =  288 bp
ORF:  95 amino acids  =  10.7 kDa
ORF:  1698 .. 2477  =  780 bp
ORF:  259 amino acids  =  29.2 kDa
ORF:  250 .. 582  =  333 bp
ORF:  110 amino acids  =  11.3 kDa
ORF:  1431 .. 2456  =  1026 bp
ORF:  341 amino acids  =  38.0 kDa
ORF:  4026 .. 4886  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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Individual Sequences & Maps

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