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pGL4.73[hRluc/SV40]

SV40 promoter-containing internal control vector for strong constitutive expression of Renilla luciferase.

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pGL4.73[hRluc SV40] Sequence and MappGL4.73[hRluc SV40].dna
Map and Sequence File   
Sequence Author:  Promega
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 BsgI (3919) AarI - BfuAI - BspMI (3891) RVprimer3 (3870 .. 3889) BsmBI (3718) SpeI (3705) BstZ17I (3386) SacII (3270) PvuI (3246) MscI (3110) AhdI (2876) BstEII (2801) BstXI - PstI (2798) AleI (2796) NotI (2774) BspHI (2674) AlwNI (2370) BaeGI - Bme1580I (2272) ApaLI (2268) BciVI (2157) DrdI (2062) BglI - SfiI (8) Bpu10I (19) Eco53kI (23) SacI (25) NheI (27) BmtI (31) AbsI - PaeR7I - PspXI - XhoI (33) BglII (45) FspI (52) PvuII (124) SexAI * (215) BglI - SfiI (401) StuI (447) AvrII (448) HindIII (464) BglI (499) DraIII (687) PfoI * (779) XmnI (1033) BsaI (1039) NruI (1160) ZraI (1194) PflFI - Tth111I (1195) AatII (1196) PasI (1273) XbaI (1436) FseI (1455) ApoI (1541) AanI - AanI - PsiI (1576) HpaI (1596) MfeI (1605) BamHI (1698) SalI (1704) PshAI (1769) RVprimer4 (1755 .. 1774) BbsI (1783) AfeI (1830) BspQI - SapI (1838) AflIII - PciI (1954) pGL4.73[hRluc/SV40] 3921 bp
BsgI  (3919)
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).
AarI  (3891)
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  (3891)
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  (3891)
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  (3718)
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  (3705)
1 site
A C T A G T T G A T C A
BstZ17I  (3386)
1 site
G T A T A C C A T A T G
SacII  (3270)
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  (3246)
1 site
C G A T C G G C T A G C
MscI  (3110)
1 site
T G G C C A A C C G G T
AhdI  (2876)
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.
BstEII  (2801)
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  (2798)
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  (2798)
1 site
C T G C A G G A C G T C
AleI  (2796)
1 site
C A C N N N N G T G G T G N N N N C A C
NotI  (2774)
1 site
G C G G C C G C C G C C G G C G
BspHI  (2674)
1 site
T C A T G A A G T A C T
AlwNI  (2370)
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.
BaeGI  (2272)
1 site
G K G C M C C M C G K G

Sticky ends from different BaeGI sites may not be compatible.
Bme1580I  (2272)
1 site
G K G C M C C M C G K G

Sticky ends from different Bme1580I sites may not be compatible.
ApaLI  (2268)
1 site
G T G C A C C A C G T G
BciVI  (2157)
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.
DrdI  (2062)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
BglI  (8)
3 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.
Bpu10I  (19)
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.
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
AbsI  (33)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (33)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (33)
1 site
V C T C G A G B B G A G C T C V
XhoI  (33)
1 site
C T C G A G G A G C T C
BglII  (45)
1 site
A G A T C T T C T A G A
FspI  (52)
1 site
T G C G C A A C G C G T
PvuII  (124)
1 site
C A G C T G G T C G A C
SexAI  (215)
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.
BglI  (401)
3 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  (401)
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.
StuI  (447)
1 site
A G G C C T T C C G G A
AvrII  (448)
1 site
C C T A G G G G A T C C
HindIII  (464)
1 site
A A G C T T T T C G A A
BglI  (499)
3 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.
DraIII  (687)
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.
PfoI  (779)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
XmnI  (1033)
1 site
G A A N N N N T T C C T T N N N N A A G
BsaI  (1039)
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.
NruI  (1160)
1 site
T C G C G A A G C G C T
ZraI  (1194)
1 site
G A C G T C C T G C A G
PflFI  (1195)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (1195)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
AatII  (1196)
1 site
G A C G T C C T G C A G
PasI  (1273)
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.
XbaI  (1436)
1 site
T C T A G A A G A T C T
FseI  (1455)
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.
ApoI  (1541)
1 site
R A A T T Y Y T T A A R

ApoI is typically used at 50°C, but is 50% active at 37°C.
AanI  (1576)
1 site
T T A T A A A A T A T T
AanI  (1576)
1 site
T T A T A A A A T A T T
PsiI  (1576)
1 site
T T A T A A A A T A T T
HpaI  (1596)
1 site
G T T A A C C A A T T G
MfeI  (1605)
1 site
C A A T T G G T T A A C
BamHI  (1698)
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.
SalI  (1704)
1 site
G T C G A C C A G C T G
PshAI  (1769)
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.
BbsI  (1783)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
AfeI  (1830)
1 site
A G C G C T T C G C G A
BspQI  (1838)
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  (1838)
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.
AflIII  (1954)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (1954)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
RVprimer3
20-mer  /  50% GC
1 binding site
3870 .. 3889  =  20 annealed bases
Tm  =  54°C
RVprimer4
20-mer  /  65% GC
1 binding site
1755 .. 1774  =  20 annealed bases
Tm  =  61°C
hRluc
499 .. 1434  =  936 bp
311 amino acids  =  36.0 kDa
Product: Renilla luciferase
human codon-optimized
hRluc
499 .. 1434  =  936 bp
311 amino acids  =  36.0 kDa
Product: Renilla luciferase
human codon-optimized
AmpR
2803 .. 3663  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2803 .. 3594  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2803 .. 3663  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3595 .. 3663  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2803 .. 3663  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2015 .. 2603  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2015 .. 2603  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
SV40 promoter
106 .. 463  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
106 .. 463  =  358 bp
SV40 enhancer and early promoter
SV40 poly(A) signal
1475 .. 1596  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1475 .. 1596  =  122 bp
SV40 polyadenylation signal
pause site
3830 .. 3921  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
pause site
3830 .. 3921  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
poly(A) signal
3768 .. 3816  =  49 bp
synthetic polyadenylation signal
poly(A) signal
3768 .. 3816  =  49 bp
synthetic polyadenylation signal
SV40 ori
314 .. 449  =  136 bp
SV40 origin of replication
SV40 ori
314 .. 449  =  136 bp
SV40 origin of replication
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