pIREShyg3

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

Sequence Author: Clontech (TaKaRa)

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SgrDI (5766) SspI (5649) FspI (5067) AhdI (4845) BspQI - SapI (3836) BstZ17I (3573) PaeR7I - PspXI - XhoI (3514) PsiI (3408) BsaBI * (3287) BsgI (3284) BclI * (3282) XbaI (3272) PasI (3178) BlpI (3107) BbvCI (3021) NruI (208) MluI (228) SpeI (249) CMV enhancer SnaBI (590) 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) EcoRV (964) BsiWI (967) BstXI (998) MscI (1219) SphI (1303) NsiI (1305) NotI (1313) PspOMI (1463) ApaI (1467) AvrII (1501) PmlI (1666) PaqCI (1689) BmgBI (1893) PshAI (1983) BsmBI - Esp3I (2024) AsiSI (2320) RsrII (2364) pIREShyg3 5768 bp
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
NruI  (208)
1 site
T C G C G A A G C G C T
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
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

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
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
PaqCI  (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 PaqCI recognition sequence.
Sticky ends from different PaqCI sites may not be compatible.
Cleavage can be improved with PaqCI Activator.
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.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (2024)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I 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
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.
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
2 segments
   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
2 segments
   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
ORF:  2987 .. 3280  =  294 bp
ORF:  97 amino acids  =  10.3 kDa
ORF:  1833 .. 2981  =  1149 bp
ORF:  382 amino acids  =  42.6 kDa
ORF:  4902 .. 5168  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1848 .. 2714  =  867 bp
ORF:  288 amino acids  =  32.7 kDa
ORF:  4772 .. 5632  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  973 .. 1302  =  330 bp
ORF:  109 amino acids  =  12.0 kDa
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