pGL4.74[hRluc TK]

Co-transfection vector for expressing Renilla luciferase under control of the medium-strength HSV thymidine kinase promoter.

Sequence Author: Promega

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BsgI (4235) BfuAI - BspMI - PaqCI (4207) RVprimer3 (4186 .. 4205) BsmBI - Esp3I (4034) SpeI (4021) PvuI (3562) AhdI (3192) BstEII (3117) BstXI (3114) AleI (3112) NotI (3090) BspHI (2990) ApaLI (2584) BciVI (2473) BssSI - BssSαI (2443) DrdI (2378) PciI (2270) AfeI (2146) RVprimer4 (2071 .. 2090) SfiI (8) Acc65I (14) KpnI (18) BssHII (261) TspMI - XmaI (302) SmaI (304) KasI (309) NarI (310) SfoI (311) PluTI (313) PpuMI (339) AvrII (342) BsaAI (366) PvuII (551) EcoRI (669) BstBI (673) MluI (733) HindIII (780) BstAPI - DraIII (1003) NsiI (1034) PfoI * (1095) EcoRV (1300) XmnI (1349) NruI (1476) ZraI (1510) PflFI - Tth111I (1511) AatII (1512) PasI (1589) XbaI (1752) FseI (1771) PsiI (1892) HpaI (1912) MfeI (1921) BamHI (2014) SalI (2020) PshAI (2085) pGL4.74[hRluc/TK] 4237 bp
BsgI  (4235)
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).
BfuAI  (4207)
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  (4207)
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.
PaqCI  (4207)
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.
BsmBI  (4034)
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  (4034)
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.
SpeI  (4021)
1 site
A C T A G T T G A T C A
PvuI  (3562)
1 site
C G A T C G G C T A G C
AhdI  (3192)
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  (3117)
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  (3114)
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.
AleI  (3112)
1 site
C A C N N N N G T G G T G N N N N C A C
NotI  (3090)
1 site
G C G G C C G C C G C C G G C G
BspHI  (2990)
1 site
T C A T G A A G T A C T
ApaLI  (2584)
1 site
G T G C A C C A C G T G
BciVI  (2473)
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.
BssSI  (2443)
1 site
C A C G A G G T G C T C
BssSαI  (2443)
1 site
C A C G A G G T G C T C
DrdI  (2378)
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.
PciI  (2270)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AfeI  (2146)
1 site
A G C G C T T C G C G A
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
BssHII  (261)
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.
TspMI  (302)
1 site
C C C G G G G G G C C C
XmaI  (302)
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  (304)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
KasI  (309)
1 site
G G C G C C C C G C G G
NarI  (310)
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  (311)
1 site
G G C G C C C C G C G G
PluTI  (313)
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.
PpuMI  (339)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
AvrII  (342)
1 site
C C T A G G G G A T C C
BsaAI  (366)
1 site
Y A C G T R R T G C A Y
PvuII  (551)
1 site
C A G C T G G T C G A C
EcoRI  (669)
1 site
G A A T T C C T T A A G
BstBI  (673)
1 site
T T C G A A A A G C T T
MluI  (733)
1 site
A C G C G T T G C G C A
HindIII  (780)
1 site
A A G C T T T T C G A A
BstAPI  (1003)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
DraIII  (1003)
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.
NsiI  (1034)
1 site
A T G C A T T A C G T A
PfoI  (1095)
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.
EcoRV  (1300)
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.
XmnI  (1349)
1 site
G A A N N N N T T C C T T N N N N A A G
NruI  (1476)
1 site
T C G C G A A G C G C T
ZraI  (1510)
1 site
G A C G T C C T G C A G
PflFI  (1511)
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  (1511)
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  (1512)
1 site
G A C G T C C T G C A G
PasI  (1589)
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  (1752)
1 site
T C T A G A A G A T C T
FseI  (1771)
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  (1892)
1 site
T T A T A A A A T A T T
HpaI  (1912)
1 site
G T T A A C C A A T T G
MfeI  (1921)
1 site
C A A T T G G T T A A C
BamHI  (2014)
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  (2020)
1 site
G T C G A C C A G C T G
PshAI  (2085)
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.
RVprimer3
20-mer  /  50% GC
1 binding site
4186 .. 4205  =  20 annealed bases
Tm  =  54°C
RVprimer4
20-mer  /  65% GC
1 binding site
2071 .. 2090  =  20 annealed bases
Tm  =  62°C
hRluc
815 .. 1750  =  936 bp
311 amino acids  =  36.0 kDa
Product: Renilla luciferase
human codon-optimized
hRluc
815 .. 1750  =  936 bp
311 amino acids  =  36.0 kDa
Product: Renilla luciferase
human codon-optimized
AmpR
3119 .. 3979  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3119 .. 3910  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3119 .. 3979  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3911 .. 3979  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3119 .. 3979  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
HSV TK promoter
28 .. 779  =  752 bp
herpes simplex virus thymidine kinase promoter
HSV TK promoter
28 .. 779  =  752 bp
herpes simplex virus thymidine kinase promoter
ori
2331 .. 2919  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2331 .. 2919  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
SV40 poly(A) signal
1791 .. 1912  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1791 .. 1912  =  122 bp
SV40 polyadenylation signal
pause site
4146 .. 4237  =  92 bp
RNA polymerase II transcriptional pause signal from the human α2 globin gene
pause site
4146 .. 4237  =  92 bp
RNA polymerase II transcriptional pause signal from the human α2 globin gene
poly(A) signal
4084 .. 4132  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4084 .. 4132  =  49 bp
synthetic polyadenylation signal
ORF:  352 .. 807  =  456 bp
ORF:  151 amino acids  =  17.1 kDa
ORF:  815 .. 1750  =  936 bp
ORF:  311 amino acids  =  36.0 kDa
ORF:  3249 .. 3515  =  267 bp
ORF:  88 amino acids  =  9.3 kDa
ORF:  1877 .. 2119  =  243 bp
ORF:  80 amino acids  =  9.0 kDa
ORF:  3119 .. 3979  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  114 .. 560  =  447 bp
ORF:  148 amino acids  =  16.0 kDa
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