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Plasmid Files

pGL4.29[luc2P/CRE/Hygro]

Vector with a minimal promoter and a cAMP response element for studying cell signaling using destabilized luciferase.

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pGL4.29[luc2P CRE Hygro] Sequence and MappGL4.29[luc2P CRE Hygro].dna
Map and Sequence File   
Sequence Author:  Promega
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 AarI - BfuAI - BspMI (6054) RVprimer3 (6033 .. 6052) poly(A) signal BsmBI (5881) SpeI (5868) BstZ17I (5549) SacII (5433) PvuI (5409) Bsu36I (5395) BstEII (4964) BstXI - PstI (4961) NotI (4937) AlwNI (4533) ApaLI (4431) BciVI (4320) PciI (4117) RVprimer4 (3918 .. 3937) SalI (3867) BstBI (3853) PmeI (3777) BssHII (3752) BglI - SfiI (8) Acc65I (14) KpnI (18) Eco53kI (23) SacI (25) NheI (27) BmtI (31) BglII (120) BglI - SfiI (133) HindIII (139) MreI - SgrAI (278) BsrGI (705) BbvCI (926) KasI (1162) NarI (1163) SfoI (1164) PluTI (1166) BlpI (1169) DraIII (1361) BpmI (1595) EcoRI (1864) FseI (2019) PsiI (2140) MfeI (2169) BamHI (2262) SV40 promoter StuI (2695) AvrII (2696) XmnI (2779) BsaAI (2927) pGL4.29[luc2P/CRE/Hygro] 6084 bp
AarI  (6054)
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  (6054)
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  (6054)
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  (5881)
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  (5868)
1 site
A C T A G T T G A T C A
BstZ17I  (5549)
1 site
G T A T A C C A T A T G
SacII  (5433)
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  (5409)
1 site
C G A T C G G C T A G C
Bsu36I  (5395)
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  (4964)
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  (4961)
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  (4961)
1 site
C T G C A G G A C G T C
NotI  (4937)
1 site
G C G G C C G C C G C C G G C G
AlwNI  (4533)
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  (4431)
1 site
G T G C A C C A C G T G
BciVI  (4320)
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  (4117)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SalI  (3867)
1 site
G T C G A C C A G C T G
BstBI  (3853)
1 site
T T C G A A A A G C T T
PmeI  (3777)
1 site
G T T T A A A C C A A A T T T G
BssHII  (3752)
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)
2 sites
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)
2 sites
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
Eco53kI  (23)
1 site
G A G C T C C T C G A G
SacI  (25)
1 site
G A G C T C C T C G A G
NheI  (27)
1 site
G C T A G C C G A T C G
BmtI  (31)
1 site
G C T A G C C G A T C G
BglII  (120)
1 site
A G A T C T T C T A G A
BglI  (133)
2 sites
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  (133)
2 sites
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.
HindIII  (139)
1 site
A A G C T T T T C G A A
MreI  (278)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (278)
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  (705)
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  (926)
1 site
C C T C A G C G G A G T C G
KasI  (1162)
1 site
G G C G C C C C G C G G
NarI  (1163)
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  (1164)
1 site
G G C G C C C C G C G G
PluTI  (1166)
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  (1169)
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  (1361)
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  (1595)
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.
EcoRI  (1864)
1 site
G A A T T C C T T A A G
FseI  (2019)
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  (2140)
1 site
T T A T A A A A T A T T
MfeI  (2169)
1 site
C A A T T G G T T A A C
BamHI  (2262)
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  (2695)
1 site
A G G C C T T C C G G A
AvrII  (2696)
1 site
C C T A G G G G A T C C
XmnI  (2779)
1 site
G A A N N N N T T C C T T N N N N A A G
BsaAI  (2927)
1 site
Y A C G T R R T G C A Y
RVprimer3
20-mer  /  50% GC
1 binding site
6033 .. 6052  =  20 annealed bases
Tm  =  54°C
RVprimer4
20-mer  /  65% GC
1 binding site
3918 .. 3937  =  20 annealed bases
Tm  =  61°C
luciferase
215 .. 1864  =  1650 bp
550 amino acids  =  60.6 kDa
Product: firefly luciferase
synthetic luc2 version of the luciferase gene
luciferase
215 .. 1864  =  1650 bp
550 amino acids  =  60.6 kDa
Product: firefly luciferase
synthetic luc2 version of the luciferase gene
HygR
2742 .. 3779  =  1038 bp
345 amino acids  =  38.4 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
HygR
2742 .. 3779  =  1038 bp
345 amino acids  =  38.4 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
AmpR
4966 .. 5826  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4966 .. 5757  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4966 .. 5826  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5758 .. 5826  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4966 .. 5826  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
4178 .. 4766  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4178 .. 4766  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
SV40 promoter
2354 .. 2711  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
2354 .. 2711  =  358 bp
SV40 enhancer and early promoter
SV40 poly(A) signal
2039 .. 2160  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2039 .. 2160  =  122 bp
SV40 polyadenylation signal
hPEST
1868 .. 1987  =  120 bp
40 amino acids  =  4.2 kDa
Product: PEST degradation sequence from mouse
ornithine decarboxylase
human codon-optimized
hPEST
1868 .. 1987  =  120 bp
40 amino acids  =  4.2 kDa
Product: PEST degradation sequence from mouse
ornithine decarboxylase
human codon-optimized
pause site
5993 .. 6084  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
pause site
5993 .. 6084  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
CRE
44 .. 109  =  66 bp
cyclic AMP response element (3 copies)
CRE
44 .. 109  =  66 bp
cyclic AMP response element (3 copies)
poly(A) signal
3803 .. 3851  =  49 bp
synthetic polyadenylation signal
poly(A) signal
3803 .. 3851  =  49 bp
synthetic polyadenylation signal
poly(A) signal
5931 .. 5979  =  49 bp
synthetic polyadenylation signal
poly(A) signal
5931 .. 5979  =  49 bp
synthetic polyadenylation signal
minP
151 .. 182  =  32 bp
minimal TATA-box promoter with low basal activity
minP
151 .. 182  =  32 bp
minimal TATA-box promoter with low basal activity
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