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pIRESpuro3

Mammalian IRES-containing vector with a puromycin resistance marker for expressing two genes from the same bicistronic transcript.

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pIRESpuro3.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Clontech (TaKaRa)
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SgrDI (5169) SspI (5052) PvuI (4618) FspI (4470) AhdI (4248) PspFI (3663) BseYI (3659) DrdI (3463) BspQI - SapI (3239) BstZ17I (2976) PaeR7I - PspXI - XhoI (2917) PsiI (2811) BclI * (2685) XbaI (2675) Bpu10I (180) NruI (208) MluI (228) SpeI (249) NdeI (484) SnaBI (590) BspDI * - ClaI * (910) EcoRV (914) NheI (927) BmtI (931) AfeI (932) AflII (934) AgeI (948) BspEI (962) BstBI (969) EcoRI (971) BamHI (977) NotI (984) BstXI (1011) AleI (1242) PstI (1305) SphI (1316) NsiI (1318) PspOMI (1466) ApaI (1470) AvrII (1504) PmlI (1669) AarI (1692) BmgBI (1896) PflFI - Tth111I (2001) RsrII (2075) BstEII (2093) BssHII (2414) PfoI * (2434) SexAI * (2526) BstAPI (2530) pIRESpuro3 5171 bp
SgrDI  (5169)
1 site
C G T C G A C G G C A G C T G C
SspI  (5052)
1 site
A A T A T T T T A T A A
PvuI  (4618)
1 site
C G A T C G G C T A G C
FspI  (4470)
1 site
T G C G C A A C G C G T
AhdI  (4248)
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.
PspFI  (3663)
1 site
C C C A G C G G G T C G
BseYI  (3659)
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.
DrdI  (3463)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
BspQI  (3239)
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  (3239)
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.
BstZ17I  (2976)
1 site
G T A T A C C A T A T G
PaeR7I  (2917)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2917)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2917)
1 site
C T C G A G G A G C T C
PsiI  (2811)
1 site
T T A T A A A A T A T T
BclI  (2685)
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.
XbaI  (2675)
1 site
T C T A G A A G A T C T
Bpu10I  (180)
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.
NruI  (208)
1 site
T C G C G A A G C G C T
MluI  (228)
1 site
A C G C G T T G C G C A
SpeI  (249)
1 site
A C T A G T T G A T C A
NdeI  (484)
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.
SnaBI  (590)
1 site
T A C G T A A T G C A T
BspDI  (910)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (910)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
EcoRV  (914)
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.
NheI  (927)
1 site
G C T A G C C G A T C G
BmtI  (931)
1 site
G C T A G C C G A T C G
AfeI  (932)
1 site
A G C G C T T C G C G A
AflII  (934)
1 site
C T T A A G G A A T T C
AgeI  (948)
1 site
A C C G G T T G G C C A
BspEI  (962)
1 site
T C C G G A A G G C C T
BstBI  (969)
1 site
T T C G A A A A G C T T
EcoRI  (971)
1 site
G A A T T C C T T A A G
BamHI  (977)
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.
NotI  (984)
1 site
G C G G C C G C C G C C G G C G
BstXI  (1011)
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.
AleI  (1242)
1 site
C A C N N N N G T G G T G N N N N C A C
PstI  (1305)
1 site
C T G C A G G A C G T C
SphI  (1316)
1 site
G C A T G C C G T A C G
NsiI  (1318)
1 site
A T G C A T T A C G T A
PspOMI  (1466)
1 site
G G G C C C C C C G G G
ApaI  (1470)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
AvrII  (1504)
1 site
C C T A G G G G A T C C
PmlI  (1669)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
AarI  (1692)
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.
BmgBI  (1896)
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.
PflFI  (2001)
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  (2001)
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.
RsrII  (2075)
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.
BstEII  (2093)
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.
BssHII  (2414)
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.
PfoI  (2434)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
SexAI  (2526)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
BstAPI  (2530)
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.
AmpR
4175 .. 5035  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4175 .. 4966  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4175 .. 5035  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4967 .. 5035  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4175 .. 5035  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
PuroR
1959 .. 2558  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
1959 .. 2558  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
3416 .. 4004  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3416 .. 4004  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
IRES
1353 .. 1926  =  574 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES
1353 .. 1926  =  574 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
chimeric intron
1051 .. 1280  =  230 bp
chimera between introns from adenovirus and immunoglobulin heavy chain genes
chimeric intron
1051 .. 1280  =  230 bp
chimera between introns from adenovirus and immunoglobulin heavy chain genes
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 poly(A) signal
2792 .. 2913  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2792 .. 2913  =  122 bp
SV40 polyadenylation signal
AmpR promoter
5036 .. 5140  =  105 bp
AmpR promoter
5036 .. 5140  =  105 bp
MCS
912 .. 1015  =  104 bp
multiple cloning site
MCS
912 .. 1015  =  104 bp
multiple cloning site
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ORF:  1836 .. 2558  =  723 bp
ORF:  240 amino acids  =  26.2 kDa
ORF:  4305 .. 4571  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1851 .. 2609  =  759 bp
ORF:  252 amino acids  =  26.3 kDa
ORF:  935 .. 1315  =  381 bp
ORF:  126 amino acids  =  13.8 kDa
ORF:  2468 .. 2701  =  234 bp
ORF:  77 amino acids  =  8.3 kDa
ORF:  4175 .. 5035  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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