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Plasmid Files

pIRES2-AcGFP1

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

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pIRES2-AcGFP1 Sequence and MappIRES2-AcGFP1.dna
Map and Sequence File   
Sequence Author:  Clontech
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 ApaLI (4935) BsaI (4320) RsrII (3847) PflFI - Tth111I (3449) FspI (3433) BspDI * - ClaI * (3171) StuI (3152) SfiI (3106) SexAI * (2920) 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) AarI (1006) BmgBI (1210) BstXI (1253) AleI (1434) BstEII (1436) Bpu10I (1446) BssHII (1580) EcoNI (1878) PpuMI (1883) NotI (1975) XbaI * (1985) MfeI (2081) HpaI (2094) AflII (2213) pIRES2-AcGFP1 5307 bp
ApaLI  (4935)
1 site
G T G C A C C A C G T G
BsaI  (4320)
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  (3847)
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  (3449)
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  (3449)
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  (3433)
1 site
T G C G C A A C G C G T
BspDI  (3171)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (3171)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
StuI  (3152)
1 site
A G G C C T T C C G G A
SfiI  (3106)
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  (2920)
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

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
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

PmlI gradually loses activity when stored at -20°C.
AarI  (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 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  (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.
AleI  (1434)
1 site
C A C N N N N G T G G T G N N N N C A C
BstEII  (1436)
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.
Bpu10I  (1446)
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.
BssHII  (1580)
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.
EcoNI  (1878)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
PpuMI  (1883)
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  (1975)
1 site
G C G G C C G C C G C C G G C G
XbaI  (1985)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
MfeI  (2081)
1 site
C A A T T G G T T A A C
HpaI  (2094)
1 site
G T T A A C C A A T T G
AflII  (2213)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
NeoR/KanR
3203 .. 3997  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
NeoR/KanR
3203 .. 3997  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
AcGFP1
1254 .. 1973  =  720 bp
239 amino acids  =  26.9 kDa
Product: Aequorea coerulescens GFP
mammalian codon-optimized
AcGFP1
1254 .. 1973  =  720 bp
239 amino acids  =  26.9 kDa
Product: Aequorea coerulescens GFP
mammalian codon-optimized
ori
4605 .. 5193  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4605 .. 5193  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES2
667 .. 1253  =  587 bp
   Segment 1:  
   667 .. 1241  =  575 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES2
667 .. 1253  =  587 bp
   Segment 2:  ATG  
   1242 .. 1244  =  3 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES2
667 .. 1253  =  587 bp
   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)
f1 ori
2223 .. 2678  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2223 .. 2678  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 promoter
2811 .. 3168  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
2811 .. 3168  =  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
2095 .. 2216  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2095 .. 2216  =  122 bp
SV40 polyadenylation signal
AmpR promoter
2705 .. 2809  =  105 bp
AmpR promoter
2705 .. 2809  =  105 bp
MCS
591 .. 665  =  75 bp
multiple cloning site
MCS
591 .. 665  =  75 bp
multiple cloning site
HSV TK poly(A) signal
4229 .. 4276  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
4229 .. 4276  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
SV40 ori
3019 .. 3154  =  136 bp
SV40 origin of replication
SV40 ori
3019 .. 3154  =  136 bp
SV40 origin of replication
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
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