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Plasmid Files

pIRESbleo3

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

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pIRESbleo3.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Clontech (TaKaRa)
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SspI (4768) PvuI (4334) FspI (4186) BpmI (4034) AhdI (3964) DrdI (3179) BspQI - SapI (2955) BstZ17I (2692) PsiI (2528) HpaI (2508) Bpu10I (180) NruI (208) MluI (228) NdeI (484) SnaBI (590) BspDI * - ClaI * (910) EcoRV (914) NotI (920) EcoRI (934) BamHI (943) BstXI (970) SacII (1100) AleI (1201) SphI (1275) NsiI (1277) PspOMI (1425) ApaI (1429) AvrII (1463) PmlI (1628) AarI (1651) KasI (1914) NarI (1915) SfoI (1916) PluTI (1918) BssHII - MauBI (1994) TspMI - XmaI (2046) SmaI (2048) SgrAI (2072) SexAI * (2121) FseI (2234) RsrII (2354) XbaI (2392) pIRESbleo3 4887 bp
SspI  (4768)
1 site
A A T A T T T T A T A A
PvuI  (4334)
1 site
C G A T C G G C T A G C
FspI  (4186)
1 site
T G C G C A A C G C G T
BpmI  (4034)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
AhdI  (3964)
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.
DrdI  (3179)
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  (2955)
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  (2955)
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  (2692)
1 site
G T A T A C C A T A T G
PsiI  (2528)
1 site
T T A T A A A A T A T T
HpaI  (2508)
1 site
G T T A A C C A A T T G
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
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.
NotI  (920)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (934)
1 site
G A A T T C C T T A A G
BamHI  (943)
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.
BstXI  (970)
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.
SacII  (1100)
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.
AleI  (1201)
1 site
C A C N N N N G T G G T G N N N N C A C
SphI  (1275)
1 site
G C A T G C C G T A C G
NsiI  (1277)
1 site
A T G C A T T A C G T A
PspOMI  (1425)
1 site
G G G C C C C C C G G G
ApaI  (1429)
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  (1463)
1 site
C C T A G G G G A T C C
PmlI  (1628)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
AarI  (1651)
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.
KasI  (1914)
1 site
G G C G C C C C G C G G
NarI  (1915)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI recognition sequence.
SfoI  (1916)
1 site
G G C G C C C C G C G G
PluTI  (1918)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
BssHII  (1994)
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.
MauBI  (1994)
1 site
C G C G C G C G G C G C G C G C
TspMI  (2046)
1 site
C C C G G G G G G C C C
XmaI  (2046)
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  (2048)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SgrAI  (2072)
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.
SexAI  (2121)
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.
FseI  (2234)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
RsrII  (2354)
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.
XbaI  (2392)
1 site
T C T A G A A G A T C T
AmpR
3891 .. 4751  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3891 .. 4682  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3891 .. 4751  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4683 .. 4751  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3891 .. 4751  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3132 .. 3720  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3132 .. 3720  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
IRES
1312 .. 1885  =  574 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES
1312 .. 1885  =  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
BleoR
1958 .. 2332  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and Zeocin™
BleoR
1958 .. 2332  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and Zeocin™
chimeric intron
1010 .. 1239  =  230 bp
chimera between introns from adenovirus and immunoglobulin heavy chain genes
chimeric intron
1010 .. 1239  =  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
2509 .. 2630  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2509 .. 2630  =  122 bp
SV40 polyadenylation signal
AmpR promoter
4752 .. 4856  =  105 bp
AmpR promoter
4752 .. 4856  =  105 bp
MCS
912 .. 974  =  63 bp
multiple cloning site
MCS
912 .. 974  =  63 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:  1795 .. 2400  =  606 bp
ORF:  201 amino acids  =  21.7 kDa
ORF:  4021 .. 4287  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1958 .. 2332  =  375 bp
ORF:  124 amino acids  =  13.8 kDa
ORF:  2492 .. 2800  =  309 bp
ORF:  102 amino acids  =  11.7 kDa
ORF:  1810 .. 2418  =  609 bp
ORF:  202 amino acids  =  21.7 kDa
ORF:  825 .. 1274  =  450 bp
ORF:  149 amino acids  =  16.0 kDa
ORF:  2106 .. 2408  =  303 bp
ORF:  100 amino acids  =  11.0 kDa
ORF:  3891 .. 4751  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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