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Plasmid Files

pNL3.2.NF-κB-RE[NlucP/NF-κB-RE/Hygro]

Vector with a minimal promoter and NF-κB response element for studying cell signaling using destabilized NanoLuc® luciferase.

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 AarI - BfuAI - BspMI (4882) RVprimer3 (4861 .. 4880) poly(A) signal BsmBI (4709) SpeI (4696) BsaHI (4404) BstZ17I (4377) SacII (4261) PvuI (4237) BstEII (3792) BstXI - PstI (3789) AleI (3787) NotI (3765) BspHI (3665) AlwNI (3361) ApaLI (3259) BciVI (3148) PciI (2945) RVprimer4 (2746 .. 2765) SalI (2695) BstBI (2681) AgeI (2616) PmeI (2605) BglI - SfiI (8) Acc65I (14) KpnI (18) Eco53kI (23) SacI (25) NheI (27) BmtI (31) BglII (85) BglI - SfiI (98) HindIII (104) PflFI - Tth111I (243) EcoNI (252) PpuMI - SanDI (571) EcoRI (692) FseI (847) PsiI (968) HpaI (988) MfeI (997) BamHI (1090) PvuII (1200) SexAI * (1291) BseRI (1520) StuI (1523) AvrII (1524) BsaAI (1755) PspOMI (1978) ApaI (1982) BssHII (2580) pNL3.2.NF- κ B-RE[NlucP/NF- κ B-RE/Hygro] 4912 bp
AarI  (4882)
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  (4882)
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  (4882)
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  (4709)
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  (4696)
1 site
A C T A G T T G A T C A
BsaHI  (4404)
1 site
G R C G Y C C Y G C R G

BsaHI is typically used at 37°C, but is even more active at 60°C.
BstZ17I  (4377)
1 site
G T A T A C C A T A T G
SacII  (4261)
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  (4237)
1 site
C G A T C G G C T A G C
BstEII  (3792)
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  (3789)
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  (3789)
1 site
C T G C A G G A C G T C
AleI  (3787)
1 site
C A C N N N N G T G G T G N N N N C A C
NotI  (3765)
1 site
G C G G C C G C C G C C G G C G
BspHI  (3665)
1 site
T C A T G A A G T A C T
AlwNI  (3361)
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  (3259)
1 site
G T G C A C C A C G T G
BciVI  (3148)
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  (2945)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SalI  (2695)
1 site
G T C G A C C A G C T G
BstBI  (2681)
1 site
T T C G A A A A G C T T
AgeI  (2616)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
PmeI  (2605)
1 site
G T T T A A A C C A A A T T T G
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  (85)
1 site
A G A T C T T C T A G A
BglI  (98)
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  (98)
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  (104)
1 site
A A G C T T T T C G A A
PflFI  (243)
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  (243)
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.
EcoNI  (252)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
PpuMI  (571)
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.
SanDI  (571)
1 site
G G G W C C C C C C W G G G

Sticky ends from different SanDI sites may not be compatible.
EcoRI  (692)
1 site
G A A T T C C T T A A G
FseI  (847)
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  (968)
1 site
T T A T A A A A T A T T
HpaI  (988)
1 site
G T T A A C C A A T T G
MfeI  (997)
1 site
C A A T T G G T T A A C
BamHI  (1090)
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.
PvuII  (1200)
1 site
C A G C T G G T C G A C
SexAI  (1291)
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.
BseRI  (1520)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
StuI  (1523)
1 site
A G G C C T T C C G G A
AvrII  (1524)
1 site
C C T A G G G G A T C C
BsaAI  (1755)
1 site
Y A C G T R R T G C A Y
PspOMI  (1978)
1 site
G G G C C C C C C G G G
ApaI  (1982)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BssHII  (2580)
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.
RVprimer3
20-mer  /  50% GC
1 binding site
4861 .. 4880  =  20 annealed bases
Tm  =  54°C
RVprimer4
20-mer  /  65% GC
1 binding site
2746 .. 2765  =  20 annealed bases
Tm  =  61°C
HygR
1570 .. 2607  =  1038 bp
345 amino acids  =  38.4 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
HygR
1570 .. 2607  =  1038 bp
345 amino acids  =  38.4 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
AmpR
3794 .. 4654  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3794 .. 4585  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3794 .. 4654  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4586 .. 4654  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3794 .. 4654  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3006 .. 3594  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3006 .. 3594  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
Nluc
180 .. 692  =  513 bp
171 amino acids  =  19.1 kDa
Product: NanoLuc® luciferase
human codon-optimized
Nluc
180 .. 692  =  513 bp
171 amino acids  =  19.1 kDa
Product: NanoLuc® luciferase
human codon-optimized
SV40 promoter
1182 .. 1539  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
1182 .. 1539  =  358 bp
SV40 enhancer and early promoter
SV40 poly(A) signal
867 .. 988  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
867 .. 988  =  122 bp
SV40 polyadenylation signal
hPEST
696 .. 815  =  120 bp
40 amino acids  =  4.2 kDa
Product: PEST degradation sequence from mouse
ornithine decarboxylase
human codon-optimized
hPEST
696 .. 815  =  120 bp
40 amino acids  =  4.2 kDa
Product: PEST degradation sequence from mouse
ornithine decarboxylase
human codon-optimized
pause site
4821 .. 4912  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
pause site
4821 .. 4912  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
NF-κB RE
33 .. 84  =  52 bp
NF-κB response element (5 copies)
NF-κB RE
33 .. 84  =  52 bp
NF-κB response element (5 copies)
poly(A) signal
2631 .. 2679  =  49 bp
synthetic polyadenylation signal
poly(A) signal
2631 .. 2679  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4759 .. 4807  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4759 .. 4807  =  49 bp
synthetic polyadenylation signal
minP
116 .. 147  =  32 bp
minimal TATA-box promoter with low basal activity
minP
116 .. 147  =  32 bp
minimal TATA-box promoter with low basal activity
SV40 ori
1390 .. 1525  =  136 bp
SV40 origin of replication
SV40 ori
1390 .. 1525  =  136 bp
SV40 origin of replication
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