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pIREShyg3

Mammalian IRES-containing vector with a hygromycin resistance marker for expressing two genes from the same bicistronic transcript.

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pIREShyg3 Sequence and MappIREShyg3.dna
Map and Sequence File   
Sequence Author:  Clontech
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 NruI (208) SgrDI (5766) AmpR promoter SspI (5649) FspI (5067) AhdI (4845) BspQI - SapI (3836) BstZ17I (3573) PaeR7I - PspXI - XhoI (3514) SV40 poly(A) signal PsiI (3408) BsaBI * (3287) BsgI (3284) BclI * (3282) XbaI (3272) PasI (3178) BlpI (3107) BbvCI (3021) RsrII (2364) MluI (228) SpeI (249) SnaBI (590) CMV promoter T7 promoter BsrGI (912) StuI (919) AflII (921) NgoMIV (926) NaeI (928) BssHII (930) NheI (934) BmtI (938) AfeI (939) BstBI (943) KasI (948) NarI (949) SfoI (950) PluTI - TspMI - XmaI (952) SmaI (954) BamHI (956) MCS EcoRV (964) BsiWI (967) BstXI (998) chimeric intron MscI (1219) SphI (1303) NsiI (1305) NotI (1313) PspOMI (1463) ApaI (1467) AvrII (1501) PmlI (1666) AarI (1689) BmgBI (1893) PshAI (1983) BsmBI (2024) AsiSI (2320) pIREShyg3 5768 bp
NruI  (208)
1 site
T C G C G A A G C G C T
SgrDI  (5766)
1 site
C G T C G A C G G C A G C T G C
SspI  (5649)
1 site
A A T A T T T T A T A A
FspI  (5067)
1 site
T G C G C A A C G C G T
AhdI  (4845)
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.
BspQI  (3836)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (3836)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
BstZ17I  (3573)
1 site
G T A T A C C A T A T G
PaeR7I  (3514)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (3514)
1 site
V C T C G A G B B G A G C T C V
XhoI  (3514)
1 site
C T C G A G G A G C T C
PsiI  (3408)
1 site
T T A T A A A A T A T T
BsaBI  (3287)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
BsgI  (3284)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BclI  (3282)
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.
XbaI  (3272)
1 site
T C T A G A A G A T C T
PasI  (3178)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
BlpI  (3107)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
BbvCI  (3021)
1 site
C C T C A G C G G A G T C G
RsrII  (2364)
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.
MluI  (228)
1 site
A C G C G T T G C G C A
SpeI  (249)
1 site
A C T A G T T G A T C A
SnaBI  (590)
1 site
T A C G T A A T G C A T
BsrGI  (912)
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.
StuI  (919)
1 site
A G G C C T T C C G G A
AflII  (921)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
NgoMIV  (926)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
NaeI  (928)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
BssHII  (930)
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.
NheI  (934)
1 site
G C T A G C C G A T C G
BmtI  (938)
1 site
G C T A G C C G A T C G
AfeI  (939)
1 site
A G C G C T T C G C G A
BstBI  (943)
1 site
T T C G A A A A G C T T
KasI  (948)
1 site
G G C G C C C C G C G G
NarI  (949)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (950)
1 site
G G C G C C C C G C G G
PluTI  (952)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
TspMI  (952)
1 site
C C C G G G G G G C C C
XmaI  (952)
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  (954)
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  (956)
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.
EcoRV  (964)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
BsiWI  (967)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
BstXI  (998)
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.
MscI  (1219)
1 site
T G G C C A A C C G G T
SphI  (1303)
1 site
G C A T G C C G T A C G
NsiI  (1305)
1 site
A T G C A T T A C G T A
NotI  (1313)
1 site
G C G G C C G C C G C C G G C G
PspOMI  (1463)
1 site
G G G C C C C C C G G G
ApaI  (1467)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
AvrII  (1501)
1 site
C C T A G G G G A T C C
PmlI  (1666)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
AarI  (1689)
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  (1893)
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.
PshAI  (1983)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BsmBI  (2024)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
AsiSI  (2320)
1 site
G C G A T C G C C G C T A G C G
HygR
1962 .. 2981  =  1020 bp
339 amino acids  =  37.8 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
HygR
1962 .. 2981  =  1020 bp
339 amino acids  =  37.8 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
AmpR
4772 .. 5632  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4772 .. 5563  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4772 .. 5632  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5564 .. 5632  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4772 .. 5632  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
4013 .. 4601  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4013 .. 4601  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES
1350 .. 1923  =  574 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES
1350 .. 1923  =  574 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
chimeric intron
1038 .. 1267  =  230 bp
chimera between introns from adenovirus and
immunoglobulin heavy chain genes
chimeric intron
1038 .. 1267  =  230 bp
chimera between introns from adenovirus and
immunoglobulin heavy chain genes
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
3389 .. 3510  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3389 .. 3510  =  122 bp
SV40 polyadenylation signal
AmpR promoter
5633 .. 5737  =  105 bp
AmpR promoter
5633 .. 5737  =  105 bp
MCS
912 .. 1002  =  91 bp
multiple cloning site
MCS
912 .. 1002  =  91 bp
multiple cloning site
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
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