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pGL4.35[luc2P/9XGAL4UAS/Hygro]

Vector with a promoter and an extended Gal4 upstream activating sequence for studying cell signaling using destabilized luciferase.

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pGL4.35[luc2P 9XGAL4UAS Hygro] Sequence and MappGL4.35[luc2P 9XGAL4UAS Hygro].dna
Map and Sequence File   
Sequence Author:  Promega
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 AarI - BfuAI - BspMI (6154) RVprimer3 (6133 .. 6152) BsmBI (5981) SpeI (5968) BstZ17I (5649) SacII (5533) PvuI (5509) Bsu36I (5495) BstEII (5064) BstXI - PstI (5061) NotI (5037) AlwNI (4633) ApaLI (4531) BciVI (4420) PciI (4217) RVprimer4 (4018 .. 4037) SalI (3967) BstBI (3953) PmeI (3877) BssHII (3852) BglI - SfiI (8) Acc65I (14) KpnI (18) PaeR7I - PspXI - XhoI (68) BspEI (192) NheI (216) BmtI (220) Ad promoter BglII (263) HindIII (280) MreI - SgrAI (378) BsrGI (805) BbvCI (1026) KasI (1262) NarI (1263) SfoI (1264) PluTI (1266) BlpI (1269) DraIII (1461) BpmI (1695) FseI (2119) PsiI (2240) MfeI (2269) BamHI (2362) SV40 promoter StuI (2795) AvrII (2796) XmnI (2879) BsaAI (3027) pGL4.35[luc2P/9XGAL4UAS/Hygro] 6184 bp
AarI  (6154)
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.
BfuAI  (6154)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (6154)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
BsmBI  (5981)
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.
SpeI  (5968)
1 site
A C T A G T T G A T C A
BstZ17I  (5649)
1 site
G T A T A C C A T A T G
SacII  (5533)
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.
PvuI  (5509)
1 site
C G A T C G G C T A G C
Bsu36I  (5495)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BstEII  (5064)
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.
BstXI  (5061)
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.
PstI  (5061)
1 site
C T G C A G G A C G T C
NotI  (5037)
1 site
G C G G C C G C C G C C G G C G
AlwNI  (4633)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
ApaLI  (4531)
1 site
G T G C A C C A C G T G
BciVI  (4420)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
PciI  (4217)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SalI  (3967)
1 site
G T C G A C C A G C T G
BstBI  (3953)
1 site
T T C G A A A A G C T T
PmeI  (3877)
1 site
G T T T A A A C C A A A T T T G
BssHII  (3852)
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.
BglI  (8)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
SfiI  (8)
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.
Acc65I  (14)
1 site
G G T A C C C C A T G G
KpnI  (18)
1 site
G G T A C C C C A T G G
PaeR7I  (68)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (68)
1 site
V C T C G A G B B G A G C T C V
XhoI  (68)
1 site
C T C G A G G A G C T C
BspEI  (192)
1 site
T C C G G A A G G C C T
NheI  (216)
1 site
G C T A G C C G A T C G
BmtI  (220)
1 site
G C T A G C C G A T C G
BglII  (263)
1 site
A G A T C T T C T A G A
HindIII  (280)
1 site
A A G C T T T T C G A A
MreI  (378)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (378)
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.
BsrGI  (805)
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.
BbvCI  (1026)
1 site
C C T C A G C G G A G T C G
KasI  (1262)
1 site
G G C G C C C C G C G G
NarI  (1263)
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  (1264)
1 site
G G C G C C C C G C G G
PluTI  (1266)
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.
BlpI  (1269)
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.
DraIII  (1461)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
BpmI  (1695)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI
recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its
electrophoretic mobility.
BpmI quickly loses activity at 37°C.
FseI  (2119)
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.
PsiI  (2240)
1 site
T T A T A A A A T A T T
MfeI  (2269)
1 site
C A A T T G G T T A A C
BamHI  (2362)
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.
StuI  (2795)
1 site
A G G C C T T C C G G A
AvrII  (2796)
1 site
C C T A G G G G A T C C
XmnI  (2879)
1 site
G A A N N N N T T C C T T N N N N A A G
BsaAI  (3027)
1 site
Y A C G T R R T G C A Y
RVprimer3
20-mer  /  50% GC
1 binding site
6133 .. 6152  =  20 annealed bases
Tm  =  54°C
RVprimer4
20-mer  /  65% GC
1 binding site
4018 .. 4037  =  20 annealed bases
Tm  =  61°C
luciferase
315 .. 1964  =  1650 bp
550 amino acids  =  60.6 kDa
Product: firefly luciferase
synthetic luc2 version of the luciferase gene
luciferase
315 .. 1964  =  1650 bp
550 amino acids  =  60.6 kDa
Product: firefly luciferase
synthetic luc2 version of the luciferase gene
HygR
2842 .. 3879  =  1038 bp
345 amino acids  =  38.4 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
HygR
2842 .. 3879  =  1038 bp
345 amino acids  =  38.4 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
AmpR
5066 .. 5926  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5066 .. 5857  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5066 .. 5926  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5858 .. 5926  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5066 .. 5926  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
4278 .. 4866  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4278 .. 4866  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
SV40 promoter
2454 .. 2811  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
2454 .. 2811  =  358 bp
SV40 enhancer and early promoter
9X GAL4 UAS
31 .. 212  =  182 bp
upstream activating sequence mediating
Gal4-dependent induction
9X GAL4 UAS
31 .. 212  =  182 bp
upstream activating sequence mediating
Gal4-dependent induction
SV40 poly(A) signal
2139 .. 2260  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2139 .. 2260  =  122 bp
SV40 polyadenylation signal
hPEST
1968 .. 2087  =  120 bp
40 amino acids  =  4.2 kDa
Product: PEST degradation sequence from mouse
ornithine decarboxylase
human codon-optimized
hPEST
1968 .. 2087  =  120 bp
40 amino acids  =  4.2 kDa
Product: PEST degradation sequence from mouse
ornithine decarboxylase
human codon-optimized
pause site
6093 .. 6184  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
pause site
6093 .. 6184  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
poly(A) signal
3903 .. 3951  =  49 bp
synthetic polyadenylation signal
poly(A) signal
3903 .. 3951  =  49 bp
synthetic polyadenylation signal
poly(A) signal
6031 .. 6079  =  49 bp
synthetic polyadenylation signal
poly(A) signal
6031 .. 6079  =  49 bp
synthetic polyadenylation signal
Ad promoter
222 .. 262  =  41 bp
adenovirus major late promoter
Ad promoter
222 .. 262  =  41 bp
adenovirus major late promoter
SV40 ori
2662 .. 2797  =  136 bp
SV40 origin of replication
SV40 ori
2662 .. 2797  =  136 bp
SV40 origin of replication
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