pIRES2-EGFP

IRES-containing bicistronic vector for expressing a gene together with EGFP.

Sequence Author: Clontech (TaKaRa)

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BsaI (4321) RsrII (3848) PflFI - Tth111I (3450) FspI (3434) PluTI (3335) SfoI (3333) NarI (3332) KasI (3331) BspDI * - ClaI * (3172) StuI (3153) SfiI (3107) CsiI - SexAI * (2921) AseI (7) NdeI (234) SnaBI (340) NheI (591) BmtI (595) AfeI (596) BglII (609) PaeR7I - XhoI (613) Eco53kI (618) SacI (620) EcoRI (629) PstI (638) SalI (639) AccI (640) SacII (652) TspMI - XmaI (656) SmaI (658) BamHI (660) AclI (690) XmnI (879) PmlI (983) PaqCI (1006) PflMI (1120) BmgBI (1210) BstXI (1253) BsrGI (1963) NotI (1976) XbaI * (1986) MfeI (2082) HpaI (2095) AflII (2214) pIRES2-EGFP 5308 bp
BsaI  (4321)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
RsrII  (3848)
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.
PflFI  (3450)
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  (3450)
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.
FspI  (3434)
1 site
T G C G C A A C G C G T
PluTI  (3335)
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.
SfoI  (3333)
1 site
G G C G C C C C G C G G
NarI  (3332)
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.
KasI  (3331)
1 site
G G C G C C C C G C G G
BspDI  (3172)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (3172)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
StuI  (3153)
1 site
A G G C C T T C C G G A
SfiI  (3107)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
CsiI  (2921)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
SexAI  (2921)
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.
AseI  (7)
1 site
A T T A A T T A A T T A
NdeI  (234)
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  (340)
1 site
T A C G T A A T G C A T
NheI  (591)
1 site
G C T A G C C G A T C G
BmtI  (595)
1 site
G C T A G C C G A T C G
AfeI  (596)
1 site
A G C G C T T C G C G A
BglII  (609)
1 site
A G A T C T T C T A G A
PaeR7I  (613)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (613)
1 site
C T C G A G G A G C T C
Eco53kI  (618)
1 site
G A G C T C C T C G A G
SacI  (620)
1 site
G A G C T C C T C G A G
EcoRI  (629)
1 site
G A A T T C C T T A A G
PstI  (638)
1 site
C T G C A G G A C G T C
SalI  (639)
1 site
G T C G A C C A G C T G
AccI  (640)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
SacII  (652)
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.
TspMI  (656)
1 site
C C C G G G G G G C C C
XmaI  (656)
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.
SmaI  (658)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BamHI  (660)
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.
AclI  (690)
1 site
A A C G T T T T G C A A
XmnI  (879)
1 site
G A A N N N N T T C C T T N N N N A A G
PmlI  (983)
1 site
C A C G T G G T G C A C
PaqCI  (1006)
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 PaqCI recognition sequence.
Sticky ends from different PaqCI sites may not be compatible.
Cleavage can be improved with PaqCI Activator.
PflMI  (1120)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
BmgBI  (1210)
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.
BstXI  (1253)
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.
BsrGI  (1963)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
NotI  (1976)
1 site
G C G G C C G C C G C C G G C G
XbaI  (1986)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
MfeI  (2082)
1 site
C A A T T G G T T A A C
HpaI  (2095)
1 site
G T T A A C C A A T T G
AflII  (2214)
1 site
C T T A A G G A A T T C
NeoR/KanR
3204 .. 3998  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
NeoR/KanR
3204 .. 3998  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
ATG
1242 .. 1244  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon for translation from IRES2
ATG
1242 .. 1244  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon for translation from IRES2
EGFP
1254 .. 1973  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
   Segment 1:  
   1254 .. 1256  =  3 bp
   1 amino acid  =  149.2 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
1254 .. 1973  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
   Segment 2:  1a  
   1257 .. 1259  =  3 bp
   1 amino acid  =  117.1 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
1254 .. 1973  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
   Segment 3:  
   1260 .. 1973  =  714 bp
   237 amino acids  =  26.7 kDa
Product: enhanced GFP
mammalian codon-optimized
EGFP
1254 .. 1973  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
Product: enhanced GFP
mammalian codon-optimized
ori
4606 .. 5194  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4606 .. 5194  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
2224 .. 2679  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2224 .. 2679  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
SV40 promoter
2812 .. 3169  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
2812 .. 3169  =  358 bp
SV40 enhancer and early promoter
CMV enhancer
61 .. 364  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
61 .. 364  =  304 bp
human cytomegalovirus immediate early enhancer
CMV promoter
365 .. 568  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
365 .. 568  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 poly(A) signal
2096 .. 2217  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2096 .. 2217  =  122 bp
SV40 polyadenylation signal
AmpR promoter
2706 .. 2810  =  105 bp
AmpR promoter
2706 .. 2810  =  105 bp
MCS
591 .. 665  =  75 bp
multiple cloning site of fluorescent protein plasmids
MCS
591 .. 665  =  75 bp
multiple cloning site of fluorescent protein plasmids
HSV TK poly(A) signal
4230 .. 4277  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
4230 .. 4277  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
IRES2
667 .. 1253  =  587 bp
3 segments
   Segment 1:  
   667 .. 1241  =  575 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES2
667 .. 1253  =  587 bp
3 segments
   Segment 2:  ATG  
   1242 .. 1244  =  3 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES2
667 .. 1253  =  587 bp
3 segments
   Segment 3:  
   1245 .. 1253  =  9 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES2
667 .. 1253  =  587 bp
3 segments
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
SV40 ori
3020 .. 3155  =  136 bp
SV40 origin of replication
SV40 ori
3020 .. 3155  =  136 bp
SV40 origin of replication
ORF:  3376 .. 3762  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  4019 .. 4468  =  450 bp
ORF:  149 amino acids  =  16.3 kDa
ORF:  1242 .. 1973  =  732 bp
ORF:  243 amino acids  =  27.3 kDa
ORF:  3204 .. 3998  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  3513 .. 4049  =  537 bp
ORF:  178 amino acids  =  19.8 kDa
ORF:  4224 .. 4457  =  234 bp
ORF:  77 amino acids  =  8.6 kDa
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