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pEF1α-IRES-DsRed-Express2

IRES-containing bicistronic vector for expressing a gene together with the DsRed-Express2 fluorescent protein.

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pEF1alpha-IRES-DsRed-Express2 Sequence and MappEF1alpha-IRES-DsRed-Express2.dna
Map and Sequence File   
Sequence Author:  Clontech
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 AseI (7) BsaI (5035) PfoI (4821) RsrII (4562) PflFI - Tth111I (4164) FspI (4148) BspDI * - ClaI * (3886) SfiI (3821) Bts α I (2885) AgeI (242) TatI (291) FseI (872) BbvCI - Bpu10I (1070) NheI (1348) BmtI (1352) AfeI (1353) EcoRI (1386) SalI (1396) AccI (1397) TspMI - XmaI (1413) SmaI (1415) BamHI (1417) BmgBI (1967) AhdI (2190) SbfI (2351) BssHII (2655) NotI (2690) XbaI * (2700) HpaI (2809) pEF1 α- IRES-DsRed-Express2 6022 bp
AseI  (7)
1 site
A T T A A T T A A T T A
BsaI  (5035)
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.
PfoI  (4821)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
RsrII  (4562)
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  (4164)
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  (4164)
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  (4148)
1 site
T G C G C A A C G C G T
BspDI  (3886)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (3886)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SfiI  (3821)
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.
BtsαI  (2885)
1 site
G C A G T G N N C G T C A C

Sticky ends from different BtsI sites may not be compatible.
AgeI  (242)
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.
TatI  (291)
1 site
W G T A C W W C A T G W
FseI  (872)
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.
BbvCI  (1070)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1070)
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.
NheI  (1348)
1 site
G C T A G C C G A T C G
BmtI  (1352)
1 site
G C T A G C C G A T C G
AfeI  (1353)
1 site
A G C G C T T C G C G A
EcoRI  (1386)
1 site
G A A T T C C T T A A G
SalI  (1396)
1 site
G T C G A C C A G C T G
AccI  (1397)
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.
TspMI  (1413)
1 site
C C C G G G G G G C C C
XmaI  (1413)
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  (1415)
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  (1417)
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.
BmgBI  (1967)
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.
AhdI  (2190)
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.
SbfI  (2351)
1 site
C C T G C A G G G G A C G T C C
BssHII  (2655)
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.
NotI  (2690)
1 site
G C G G C C G C C G C C G G C G
XbaI  (2700)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
HpaI  (2809)
1 site
G T T A A C C A A T T G
EF-1α promoter
165 .. 1346  =  1182 bp
strong constitutive promoter for human elongation
factor EF-1α
EF-1α promoter
165 .. 1346  =  1182 bp
strong constitutive promoter for human elongation
factor EF-1α
NeoR/KanR
3918 .. 4712  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
NeoR/KanR
3918 .. 4712  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
DsRed-Express2
2011 .. 2688  =  678 bp
225 amino acids  =  25.7 kDa
Product: noncytotoxic tetrameric variant of DsRed
fluorescent protein
mammalian codon-optimized
DsRed-Express2
2011 .. 2688  =  678 bp
225 amino acids  =  25.7 kDa
Product: noncytotoxic tetrameric variant of DsRed
fluorescent protein
mammalian codon-optimized
ori
5320 .. 5908  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5320 .. 5908  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES2
1424 .. 2010  =  587 bp
   Segment 1:  
   1424 .. 1998  =  575 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES2
1424 .. 2010  =  587 bp
   Segment 2:  ATG  
   1999 .. 2001  =  3 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES2
1424 .. 2010  =  587 bp
   Segment 3:  
   2002 .. 2010  =  9 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES2
1424 .. 2010  =  587 bp
3 segments
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
f1 ori
2938 .. 3393  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2938 .. 3393  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 promoter
3526 .. 3883  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
3526 .. 3883  =  358 bp
SV40 enhancer and early promoter
SV40 poly(A) signal
2810 .. 2931  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2810 .. 2931  =  122 bp
SV40 polyadenylation signal
AmpR promoter
3420 .. 3524  =  105 bp
AmpR promoter
3420 .. 3524  =  105 bp
MCS
1366 .. 1422  =  57 bp
multiple cloning site of fluorescent protein plasmids
MCS
1366 .. 1422  =  57 bp
multiple cloning site of fluorescent protein plasmids
HSV TK poly(A) signal
4944 .. 4991  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
4944 .. 4991  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
EF-1α intron A
395 .. 1337  =  943 bp
intron upstream of the start codon
EF-1α intron A
395 .. 1337  =  943 bp
intron upstream of the start codon
SV40 ori
3734 .. 3869  =  136 bp
SV40 origin of replication
SV40 ori
3734 .. 3869  =  136 bp
SV40 origin of replication
ATG
1999 .. 2001  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon for translation from IRES2
ATG
1999 .. 2001  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon for translation from IRES2
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