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Plasmid Files

pEF1α-IRES-AcGFP1

IRES-containing bicistronic vector for expressing a gene together with the AcGFP1 fluorescent protein.

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pEF1alpha-IRES-AcGFP1 Sequence and MappEF1alpha-IRES-AcGFP1.dna
Map and Sequence File   
Sequence Author:  Clontech
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 AseI (7) ApaLI (5692) BsaI (5077) RsrII (4604) PflFI - Tth111I (4206) FspI (4190) BspDI * - ClaI * (3928) SfiI (3863) SexAI * (3677) HpaI (2851) AgeI (242) FseI (872) BbvCI (1070) NheI (1348) BmtI (1352) AfeI (1353) EcoRI (1386) SalI (1396) AccI (1397) TspMI - XmaI (1413) SmaI (1415) BamHI (1417) PmlI (1740) AarI (1763) BmgBI (1967) BstEII (2193) BssHII (2337) PpuMI (2640) NotI (2732) XbaI * (2742) pEF1 α- IRES-AcGFP1 6064 bp
AseI  (7)
1 site
A T T A A T T A A T T A
ApaLI  (5692)
1 site
G T G C A C C A C G T G
BsaI  (5077)
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  (4604)
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  (4206)
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  (4206)
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  (4190)
1 site
T G C G C A A C G C G T
BspDI  (3928)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (3928)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SfiI  (3863)
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.
SexAI  (3677)
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.
HpaI  (2851)
1 site
G T T A A C C A A T T G
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.
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
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.
PmlI  (1740)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
AarI  (1763)
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.
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.
BstEII  (2193)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
BssHII  (2337)
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.
PpuMI  (2640)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
NotI  (2732)
1 site
G C G G C C G C C G C C G G C G
XbaI  (2742)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
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
3960 .. 4754  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
NeoR/KanR
3960 .. 4754  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
AcGFP1
2011 .. 2730  =  720 bp
239 amino acids  =  26.9 kDa
Product: Aequorea coerulescens GFP
mammalian codon-optimized
AcGFP1
2011 .. 2730  =  720 bp
239 amino acids  =  26.9 kDa
Product: Aequorea coerulescens GFP
mammalian codon-optimized
ori
5362 .. 5950  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5362 .. 5950  =  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
2980 .. 3435  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2980 .. 3435  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 promoter
3568 .. 3925  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
3568 .. 3925  =  358 bp
SV40 enhancer and early promoter
SV40 poly(A) signal
2852 .. 2973  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2852 .. 2973  =  122 bp
SV40 polyadenylation signal
AmpR promoter
3462 .. 3566  =  105 bp
AmpR promoter
3462 .. 3566  =  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
4986 .. 5033  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
4986 .. 5033  =  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
3776 .. 3911  =  136 bp
SV40 origin of replication
SV40 ori
3776 .. 3911  =  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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