pEF1alpha-IRES-DsRed-Express2

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

Sequence Author: Clontech (TaKaRa)

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AseI (7) BsaI (5035) PfoI (4821) RsrII (4562) PflFI - Tth111I (4164) FspI (4148) BspDI * - ClaI * (3886) SfiI (3821) BtsI - BtsαI (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.
BtsI  (2885)
1 site
G C A G T G N N C G T C A C
BtsαI  (2885)
1 site
G C A G T G N N C G T C A C

Sticky ends from different BtsαI sites may not be compatible.
AgeI  (242)
1 site
A C C G G T T G G C C A
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

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
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)
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
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
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
IRES2
1424 .. 2010  =  587 bp
3 segments
   Segment 1:  
   1424 .. 1998  =  575 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES2
1424 .. 2010  =  587 bp
3 segments
   Segment 2:  ATG  
   1999 .. 2001  =  3 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES2
1424 .. 2010  =  587 bp
3 segments
   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)
SV40 ori
3734 .. 3869  =  136 bp
SV40 origin of replication
SV40 ori
3734 .. 3869  =  136 bp
SV40 origin of replication
ORF:  724 .. 1161  =  438 bp
ORF:  145 amino acids  =  15.9 kDa
ORF:  1999 .. 2688  =  690 bp
ORF:  229 amino acids  =  26.1 kDa
ORF:  4090 .. 4476  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  803 .. 1036  =  234 bp
ORF:  77 amino acids  =  7.7 kDa
ORF:  4733 .. 5182  =  450 bp
ORF:  149 amino acids  =  16.3 kDa
ORF:  3918 .. 4712  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  439 .. 1086  =  648 bp
ORF:  215 amino acids  =  23.7 kDa
ORF:  4227 .. 4763  =  537 bp
ORF:  178 amino acids  =  19.8 kDa
ORF:  4938 .. 5171  =  234 bp
ORF:  77 amino acids  =  8.6 kDa
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