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Plasmid Files

pLightSwitch_Prom

Vector for measuring promoter activity with an enhanced and destabilized Renilla luciferase (RenSP).

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pLightSwitch_Prom Sequence and MappLightSwitch_Prom.dna
Map and Sequence File   
Sequence Author:  SwitchGear Genomics
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 SfiI (8) AarI (3626) Forward Sequencing Primer (3576 .. 3598) poly(A) signal BsmBI (3453) SpeI (3440) PvuI (2981) AseI (2783) NmeAIII (2759) AhdI (2611) BstEII (2536) BstXI - PstI (2533) AleI (2531) NotI (2509) BspHI (2409) AlwNI (2105) BciVI (1892) DrdI (1797) Acc65I (14) KpnI (18) Eco53kI (23) SacI (25) MluI (29) NheI (35) MCS BmtI (39) TspMI - XmaI (40) SmaI - SrfI (42) PaeR7I - XhoI (46) BglII (50) HindIII (67) NcoI (110) Reverse Sequencing Primer (184 .. 205) DraIII (300) SphI (538) EcoRV (585) PfoI * (635) BbvCI (760) XcmI (761) PvuII (764) BssHII (843) EcoO109I (957) EcoRI (1044) XbaI (1171) FseI (1190) SV40 poly(A) signal PsiI (1311) HpaI (1331) MfeI (1340) BamHI (1433) SalI (1439) AccI (1440) PshAI (1504) PciI (1689) pLightSwitch_Prom 3656 bp
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.
AarI  (3626)
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.
BsmBI  (3453)
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  (3440)
1 site
A C T A G T T G A T C A
PvuI  (2981)
1 site
C G A T C G G C T A G C
AseI  (2783)
1 site
A T T A A T T A A T T A
NmeAIII  (2759)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
AhdI  (2611)
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  (2536)
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  (2533)
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  (2533)
1 site
C T G C A G G A C G T C
AleI  (2531)
1 site
C A C N N N N G T G G T G N N N N C A C
NotI  (2509)
1 site
G C G G C C G C C G C C G G C G
BspHI  (2409)
1 site
T C A T G A A G T A C T
AlwNI  (2105)
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.
BciVI  (1892)
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  (1797)
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.
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
MluI  (29)
1 site
A C G C G T T G C G C A
NheI  (35)
1 site
G C T A G C C G A T C G
BmtI  (39)
1 site
G C T A G C C G A T C G
TspMI  (40)
1 site
C C C G G G G G G C C C
XmaI  (40)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (42)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (42)
1 site
G C C C G G G C C G G G C C C G
PaeR7I  (46)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (46)
1 site
C T C G A G G A G C T C
BglII  (50)
1 site
A G A T C T T C T A G A
HindIII  (67)
1 site
A A G C T T T T C G A A
NcoI  (110)
1 site
C C A T G G G G T A C C
DraIII  (300)
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.
SphI  (538)
1 site
G C A T G C C G T A C G
EcoRV  (585)
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.
PfoI  (635)
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.
BbvCI  (760)
1 site
C C T C A G C G G A G T C G
XcmI  (761)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
PvuII  (764)
1 site
C A G C T G G T C G A C
BssHII  (843)
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.
EcoO109I  (957)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
EcoRI  (1044)
1 site
G A A T T C C T T A A G
XbaI  (1171)
1 site
T C T A G A A G A T C T
FseI  (1190)
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  (1311)
1 site
T T A T A A A A T A T T
HpaI  (1331)
1 site
G T T A A C C A A T T G
MfeI  (1340)
1 site
C A A T T G G T T A A C
BamHI  (1433)
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  (1439)
1 site
G T C G A C C A G C T G
AccI  (1440)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the
recognition sequence.
Sticky ends from different AccI sites may not be compatible.
PshAI  (1504)
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.
PciI  (1689)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
Forward Sequencing Primer
23-mer  /  30% GC
1 binding site
3576 .. 3598  =  23 annealed bases
Tm  =  53°C
Reverse Sequencing Primer
22-mer  /  50% GC
1 binding site
184 .. 205  =  22 annealed bases
Tm  =  61°C
Rluc
112 .. 1044  =  933 bp
311 amino acids  =  36.0 kDa
Product: enhanced Renilla luciferase
Rluc
112 .. 1044  =  933 bp
311 amino acids  =  36.0 kDa
Product: enhanced Renilla luciferase
AmpR
2538 .. 3398  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2538 .. 3329  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2538 .. 3398  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3330 .. 3398  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2538 .. 3398  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1750 .. 2338  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1750 .. 2338  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
SV40 poly(A) signal
1210 .. 1331  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1210 .. 1331  =  122 bp
SV40 polyadenylation signal
hPEST
1048 .. 1167  =  120 bp
40 amino acids  =  4.2 kDa
Product: PEST degradation sequence from mouse
ornithine decarboxylase
human codon-optimized
hPEST
1048 .. 1167  =  120 bp
40 amino acids  =  4.2 kDa
Product: PEST degradation sequence from mouse
ornithine decarboxylase
human codon-optimized
pause site
3565 .. 3656  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
pause site
3565 .. 3656  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
MCS
1 .. 72  =  72 bp
multiple cloning site
MCS
1 .. 72  =  72 bp
multiple cloning site
poly(A) signal
3503 .. 3551  =  49 bp
synthetic polyadenylation signal
poly(A) signal
3503 .. 3551  =  49 bp
synthetic polyadenylation signal
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