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pIRESneo3

Mammalian IRES-containing vector with a neomycin (G418) resistance marker for expressing two genes from the same bicistronic transcript.

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pIRESneo3 Sequence and MappIRESneo3.dna
Map and Sequence File   
Sequence Author:  Clontech
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 SgrDI (5273) SspI (5156) PvuI (4722) BpmI (4422) AhdI (4352) BstZ17I (3080) PsiI (2914) BclI * (2788) XbaI (2778) Bpu10I (180) NruI (208) SpeI (249) NdeI (484) SnaBI (590) BspDI * - ClaI * (910) EcoRV (914) NheI (927) BmtI (931) AfeI (932) AflII (934) StuI (940) AgeI (948) BsiWI (954) BspEI (962) BsmBI (964) BstBI (969) EcoRI (971) BamHI (977) NotI (984) BstXI (1011) AleI (1242) NsiI (1318) PspOMI (1466) ApaI (1470) AvrII (1504) PmlI (1669) AarI (1692) DraIII (1716) BmgBI (1896) AvaI - BsoBI - TspMI - XmaI (1938) BmeT110I (1939) SmaI (1940) KasI (2098) NarI (2099) SfoI (2100) PluTI (2102) PflFI - Tth111I (2217) BssHII (2496) NgoMIV (2599) NaeI (2601) RsrII (2615) pIRESneo3 5275 bp
SgrDI  (5273)
1 site
C G T C G A C G G C A G C T G C
SspI  (5156)
1 site
A A T A T T T T A T A A
PvuI  (4722)
1 site
C G A T C G G C T A G C
BpmI  (4422)
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  (4352)
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.
BstZ17I  (3080)
1 site
G T A T A C C A T A T G
PsiI  (2914)
1 site
T T A T A A A A T A T T
BclI  (2788)
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  (2778)
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
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

The sticky ends produced by AflII are hard to ligate.
StuI  (940)
1 site
A G G C C T T C C G G A
AgeI  (948)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BsiWI  (954)
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.
BspEI  (962)
1 site
T C C G G A A G G C C T
BsmBI  (964)
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.
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
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.
DraIII  (1716)
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.
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.
AvaI  (1938)
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  (1938)
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  (1938)
1 site
C C C G G G G G G C C C
XmaI  (1938)
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.
BmeT110I  (1939)
1 site
C Y C G R G G R G C Y C
SmaI  (1940)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
KasI  (2098)
1 site
G G C G C C C C G C G G
NarI  (2099)
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  (2100)
1 site
G G C G C C C C G C G G
PluTI  (2102)
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.
PflFI  (2217)
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  (2217)
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.
BssHII  (2496)
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.
NgoMIV  (2599)
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.
NaeI  (2601)
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.
RsrII  (2615)
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.
AmpR
4279 .. 5139  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4279 .. 5070  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4279 .. 5139  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5071 .. 5139  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4279 .. 5139  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
1962 .. 2765  =  804 bp
267 amino acids  =  29.2 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
NeoR/KanR
1962 .. 2765  =  804 bp
267 amino acids  =  29.2 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
ori
3520 .. 4108  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3520 .. 4108  =  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
2895 .. 3016  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2895 .. 3016  =  122 bp
SV40 polyadenylation signal
AmpR promoter
5140 .. 5244  =  105 bp
AmpR promoter
5140 .. 5244  =  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
M13 rev
3118 .. 3134  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
3118 .. 3134  =  17 bp
common sequencing primer, one of multiple similar
variants
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