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pNL2.3[secNluc/Hygro]

Promoterless vector for measuring the activity of promoter and enhancer sequences with a secreted NanoLuc® luciferase assay.

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pNL2.3[secNluc Hygro] Sequence and MappNL2.3[secNluc Hygro].dna
Map and Sequence File   
Sequence Author:  Promega
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 AarI - BfuAI - BspMI (4763) RVprimer3 (4742 .. 4761) poly(A) signal BsmBI (4590) SpeI (4577) BsaHI (4285) BstZ17I (4258) SacII (4142) PvuI (4118) Bsu36I (4104) BstEII (3673) BstXI - PstI (3670) AleI (3668) NotI (3646) BspHI (3546) AlwNI (3242) ApaLI (3140) BciVI (3029) PciI (2826) RVprimer4 (2627 .. 2646) SalI (2576) BstBI (2562) AgeI (2497) PmeI (2486) BssHII (2461) BglI - SfiI (8) Acc65I (14) KpnI (18) Eco53kI (23) SacI (25) NheI (27) BmtI (31) AbsI - PaeR7I - PspXI - XhoI (33) EcoRV (41) BglII (46) BglI - SfiI (59) HindIII (65) PasI (144) PflFI - Tth111I (247) EcoNI (256) PpuMI - SanDI (575) FseI (728) AanI - PsiI (849) HpaI (869) MfeI (878) BamHI (971) PvuII (1081) SexAI * (1172) BseRI (1401) StuI (1404) AvrII (1405) BsaAI (1636) PspOMI (1859) ApaI (1863) pNL2.3[secNluc/Hygro] 4793 bp
AarI  (4763)
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  (4763)
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  (4763)
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  (4590)
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  (4577)
1 site
A C T A G T T G A T C A
BsaHI  (4285)
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  (4258)
1 site
G T A T A C C A T A T G
SacII  (4142)
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  (4118)
1 site
C G A T C G G C T A G C
Bsu36I  (4104)
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  (3673)
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  (3670)
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  (3670)
1 site
C T G C A G G A C G T C
AleI  (3668)
1 site
C A C N N N N G T G G T G N N N N C A C
NotI  (3646)
1 site
G C G G C C G C C G C C G G C G
BspHI  (3546)
1 site
T C A T G A A G T A C T
AlwNI  (3242)
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  (3140)
1 site
G T G C A C C A C G T G
BciVI  (3029)
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  (2826)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SalI  (2576)
1 site
G T C G A C C A G C T G
BstBI  (2562)
1 site
T T C G A A A A G C T T
AgeI  (2497)
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  (2486)
1 site
G T T T A A A C C A A A T T T G
BssHII  (2461)
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
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
EcoRV  (41)
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.
BglII  (46)
1 site
A G A T C T T C T A G A
BglI  (59)
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  (59)
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  (65)
1 site
A A G C T T T T C G A A
PasI  (144)
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.
PflFI  (247)
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  (247)
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  (256)
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  (575)
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  (575)
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.
FseI  (728)
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.
AanI  (849)
1 site
T T A T A A A A T A T T
PsiI  (849)
1 site
T T A T A A A A T A T T
HpaI  (869)
1 site
G T T A A C C A A T T G
MfeI  (878)
1 site
C A A T T G G T T A A C
BamHI  (971)
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  (1081)
1 site
C A G C T G G T C G A C
SexAI  (1172)
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  (1401)
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  (1404)
1 site
A G G C C T T C C G G A
AvrII  (1405)
1 site
C C T A G G G G A T C C
BsaAI  (1636)
1 site
Y A C G T R R T G C A Y
PspOMI  (1859)
1 site
G G G C C C C C C G G G
ApaI  (1863)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
RVprimer3
20-mer  /  50% GC
1 binding site
4742 .. 4761  =  20 annealed bases
Tm  =  54°C
RVprimer4
20-mer  /  65% GC
1 binding site
2627 .. 2646  =  20 annealed bases
Tm  =  61°C
HygR
1451 .. 2488  =  1038 bp
345 amino acids  =  38.4 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
HygR
1451 .. 2488  =  1038 bp
345 amino acids  =  38.4 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
AmpR
3675 .. 4535  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3675 .. 4466  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3675 .. 4535  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4467 .. 4535  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3675 .. 4535  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2887 .. 3475  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2887 .. 3475  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
Nluc
187 .. 699  =  513 bp
170 amino acids  =  19.0 kDa
Product: NanoLuc® luciferase
human codon-optimized
Nluc
187 .. 699  =  513 bp
170 amino acids  =  19.0 kDa
Product: NanoLuc® luciferase
human codon-optimized
SV40 promoter
1063 .. 1420  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
1063 .. 1420  =  358 bp
SV40 enhancer and early promoter
SV40 poly(A) signal
748 .. 869  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
748 .. 869  =  122 bp
SV40 polyadenylation signal
pause site
4702 .. 4793  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
pause site
4702 .. 4793  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
MCS
1 .. 70  =  70 bp
multiple cloning site
MCS
1 .. 70  =  70 bp
multiple cloning site
poly(A) signal
2512 .. 2560  =  49 bp
synthetic polyadenylation signal
poly(A) signal
2512 .. 2560  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4640 .. 4688  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4640 .. 4688  =  49 bp
synthetic polyadenylation signal
SV40 ori
1271 .. 1406  =  136 bp
SV40 origin of replication
SV40 ori
1271 .. 1406  =  136 bp
SV40 origin of replication
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