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Plasmid Files

pPTR I

Chromosomal integrating E. coli-Aspergillus shuttle vector with the ptrA gene conferring resistance to pyrithiamine.            

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pPTR I.dna
Map and Sequence File:    Download    Open   
Sequence Author:  TaKaRa
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NgoMIV (4780) KpnI (4412) Acc65I (4408) SmaI (4406) TspMI - XmaI (4404) PstI - SbfI (4385) BfuAI - BspMI (4374) HindIII (4369) BciVI (3812) AlwNI (3601) BspHI (3290) AhdI (3122) ScaI (2641) TatI (2639) NaeI (0) BtgZI (98) BsaAI (103) DraIII (106) PsiI (231) NdeI (422) EagI (742) AfeI (807) BstEII (914) SacII (1144) BsmBI (1258) BsaBI (1285) BsiWI (1319) PflFI - Tth111I (1362) PpuMI * (1388) AgeI (2215) MfeI (2309) pPTR I 4782 bp
NgoMIV  (4780)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
KpnI  (4412)
1 site
G G T A C C C C A T G G
Acc65I  (4408)
1 site
G G T A C C C C A T G G
SmaI  (4406)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (4404)
1 site
C C C G G G G G G C C C
XmaI  (4404)
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.
PstI  (4385)
1 site
C T G C A G G A C G T C
SbfI  (4385)
1 site
C C T G C A G G G G A C G T C C
BfuAI  (4374)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (4374)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
HindIII  (4369)
1 site
A A G C T T T T C G A A
BciVI  (3812)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
AlwNI  (3601)
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.
BspHI  (3290)
1 site
T C A T G A A G T A C T
AhdI  (3122)
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.
ScaI  (2641)
1 site
A G T A C T T C A T G A
TatI  (2639)
1 site
W G T A C W W C A T G W
NaeI  (0)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI recognition sequence.
BtgZI  (98)
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.
BsaAI  (103)
1 site
Y A C G T R R T G C A Y
DraIII  (106)
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.
PsiI  (231)
1 site
T T A T A A A A T A T T
NdeI  (422)
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.
EagI  (742)
1 site
C G G C C G G C C G G C
AfeI  (807)
1 site
A G C G C T T C G C G A
BstEII  (914)
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.
SacII  (1144)
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.
BsmBI  (1258)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BsaBI  (1285)
1 site
G A T N N N N A T C C T A N N N N T A G
BsiWI  (1319)
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.
PflFI  (1362)
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  (1362)
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.
PpuMI  (1388)
1 site
R G G W C C Y Y C C W G G R
* Blocked by Dcm methylation.
Sticky ends from different PpuMI sites may not be compatible.
AgeI  (2215)
1 site
A C C G G T T G G C C A
MfeI  (2309)
1 site
C A A T T G G T T A A C
ptrA
310 .. 2317  =  2008 bp
Product: pyrithiamine resistance gene of Aspergillus oryzae (Kubodera et al., 2000)
useful as a dominant selectable marker in Aspergillus
ptrA
310 .. 2317  =  2008 bp
Product: pyrithiamine resistance gene of Aspergillus oryzae (Kubodera et al., 2000)
useful as a dominant selectable marker in Aspergillus
AmpR
2335 .. 3195  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2335 .. 2403  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2335 .. 3195  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2404 .. 3195  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2335 .. 3195  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3366 .. 3954  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3366 .. 3954  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
4655 .. 301  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4655 .. 301  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
MCS
4369 .. 4413  =  45 bp
multiple cloning site
MCS
4369 .. 4413  =  45 bp
multiple cloning site
lac promoter
4278 .. 4308  =  31 bp
   Segment 1:  -35  
   4278 .. 4283  =  6 bp
promoter for the E. coli lac operon
lac promoter
4278 .. 4308  =  31 bp
   Segment 2:  
   4284 .. 4301  =  18 bp
promoter for the E. coli lac operon
lac promoter
4278 .. 4308  =  31 bp
   Segment 3:  -10  
   4302 .. 4308  =  7 bp
promoter for the E. coli lac operon
lac promoter
4278 .. 4308  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
4316 .. 4332  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
4316 .. 4332  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
M13 rev
4340 .. 4356  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
4340 .. 4356  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
4426 .. 4442  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
4426 .. 4442  =  17 bp
common sequencing primer, one of multiple similar variants
ptrA
554 .. 1595  =  1042 bp
327 amino acids  =  35.1 kDa
2 segments
Product: pyrithiamine resistance protein (Kubodera et al., 2000)
useful as a dominant selectable marker in Aspergillus
ptrA
554 .. 1595  =  1042 bp
327 amino acids  =  35.1 kDa
   Segment 2:  
   554 .. 1200  =  647 bp
   215 amino acids  =  23.1 kDa
Product: pyrithiamine resistance protein (Kubodera et al., 2000)
useful as a dominant selectable marker in Aspergillus
ptrA
554 .. 1595  =  1042 bp
327 amino acids  =  35.1 kDa
   Segment 1:  
   1259 .. 1595  =  337 bp
   112 amino acids  =  12.0 kDa
Product: pyrithiamine resistance protein (Kubodera et al., 2000)
useful as a dominant selectable marker in Aspergillus
ptrA
554 .. 1595  =  1042 bp
327 amino acids  =  35.1 kDa
2 segments
Product: pyrithiamine resistance protein (Kubodera et al., 2000)
useful as a dominant selectable marker in Aspergillus
lacZα
4352 .. 4678  =  327 bp
108 amino acids  =  12.4 kDa
Product: LacZα fragment of β-galactosidase
lacZα
4352 .. 4678  =  327 bp
108 amino acids  =  12.4 kDa
Product: LacZα fragment of β-galactosidase
ORF:  2335 .. 3195  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  4352 .. 4678  =  327 bp
ORF:  108 amino acids  =  12.4 kDa
ORF:  321 .. 545  =  225 bp
ORF:  74 amino acids  =  8.4 kDa
ORF:  4279 .. 4635  =  357 bp
ORF:  118 amino acids  =  13.5 kDa
ORF:  990 .. 1247  =  258 bp
ORF:  85 amino acids  =  9.7 kDa
ORF:  1251 .. 1595  =  345 bp
ORF:  114 amino acids  =  12.2 kDa
ORF:  2799 .. 3065  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  554 .. 1162  =  609 bp
ORF:  202 amino acids  =  21.9 kDa
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