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pIRES2-ZsGreen1

IRES-containing bicistronic vector for expressing a gene together with the fluorescent protein ZsGreen1.

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pIRES2-ZsGreen1 Sequence and MappIRES2-ZsGreen1.dna
Map and Sequence File   
Sequence Author:  Clontech
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 ApaLI (4911) BsaI (4296) PfoI (4082) RsrII (3823) FspI (3409) BspDI * - ClaI * (3147) StuI (3128) SfiI (3082) SexAI * (2896) AseI (7) CMV enhancer NdeI (234) SnaBI (340) NheI (591) BmtI (595) AfeI (596) BglII (609) PaeR7I - XhoI (613) Eco53kI (618) SacI (620) EcoRI (629) PstI (638) SacII (652) TspMI - XmaI (656) SmaI (658) BamHI (660) AclI (690) XmnI (879) PmlI (983) AarI (1006) PflMI (1120) BstXI (1253) BclI * (1336) SgrAI (1341) AhdI (1433) BsrGI (1804) Bpu10I (1946) NotI (1951) XbaI * (1961) MfeI (2057) HpaI (2070) Bts α I (2146) AflII (2189) pIRES2-ZsGreen1 5283 bp
ApaLI  (4911)
1 site
G T G C A C C A C G T G
BsaI  (4296)
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  (4082)
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  (3823)
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.
FspI  (3409)
1 site
T G C G C A A C G C G T
BspDI  (3147)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (3147)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
StuI  (3128)
1 site
A G G C C T T C C G G A
SfiI  (3082)
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  (2896)
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
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.
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.
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.
BclI  (1336)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
SgrAI  (1341)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
AhdI  (1433)
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.
BsrGI  (1804)
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.
Bpu10I  (1946)
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.
NotI  (1951)
1 site
G C G G C C G C C G C C G G C G
XbaI  (1961)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
MfeI  (2057)
1 site
C A A T T G G T T A A C
HpaI  (2070)
1 site
G T T A A C C A A T T G
BtsαI  (2146)
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.
AflII  (2189)
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
3179 .. 3973  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
NeoR/KanR
3179 .. 3973  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
ZsGreen1
1254 .. 1949  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ZsGreen1
1254 .. 1949  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ori
4581 .. 5169  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4581 .. 5169  =  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
2199 .. 2654  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2199 .. 2654  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 promoter
2787 .. 3144  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
2787 .. 3144  =  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
2071 .. 2192  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2071 .. 2192  =  122 bp
SV40 polyadenylation signal
AmpR promoter
2681 .. 2785  =  105 bp
AmpR promoter
2681 .. 2785  =  105 bp
MCS
591 .. 665  =  75 bp
multiple cloning site
MCS
591 .. 665  =  75 bp
multiple cloning site
HSV TK poly(A) signal
4205 .. 4252  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
4205 .. 4252  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
SV40 ori
2995 .. 3130  =  136 bp
SV40 origin of replication
SV40 ori
2995 .. 3130  =  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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