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Plasmid Files

pNL1.1[Nluc]

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

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pNL1.1[Nluc] Sequence and MappNL1.1[Nluc].dna
Map and Sequence File   
Sequence Author:  Promega
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 AarI - BfuAI - BspMI (3080) RVprimer3 (3059 .. 3078) poly(A) signal BsmBI (2907) SpeI (2894) BsaHI (2602) BstZ17I (2575) SacII (2459) PvuI (2435) Bsu36I (2421) MscI (2299) AhdI (2065) BstEII (1990) BstXI - PstI (1987) AleI (1985) NotI (1963) BspHI (1863) AlwNI (1559) BglI - SfiI (8) Acc65I (14) KpnI (18) Eco53kI (23) BanII - SacI (25) NheI (27) BmtI (31) AbsI - PaeR7I - PspXI - XhoI (33) EcoRV (41) BglII (46) BglI - SfiI (59) HindIII (65) NcoI - StyI (98) PflFI - Tth111I (163) EcoNI (172) BtgZI (438) PpuMI - SanDI (491) XmnI (602) XbaI (625) FseI (644) AanI - AanI - PsiI (765) HpaI (785) MfeI (794) BamHI (887) SalI (893) PshAI (958) RVprimer4 (944 .. 963) AfeI (1019) BspQI - SapI (1027) AflIII - PciI (1143) NspI (1147) BciVI (1346) ApaLI (1457) BaeGI - Bme1580I (1461) pNL1.1[Nluc] 3110 bp
AarI  (3080)
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  (3080)
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  (3080)
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  (2907)
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  (2894)
1 site
A C T A G T T G A T C A
BsaHI  (2602)
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  (2575)
1 site
G T A T A C C A T A T G
SacII  (2459)
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  (2435)
1 site
C G A T C G G C T A G C
Bsu36I  (2421)
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.
MscI  (2299)
1 site
T G G C C A A C C G G T
AhdI  (2065)
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  (1990)
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  (1987)
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  (1987)
1 site
C T G C A G G A C G T C
AleI  (1985)
1 site
C A C N N N N G T G G T G N N N N C A C
NotI  (1963)
1 site
G C G G C C G C C G C C G G C G
BspHI  (1863)
1 site
T C A T G A A G T A C T
AlwNI  (1559)
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.
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
BanII  (25)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
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
NcoI  (98)
1 site
C C A T G G G G T A C C
StyI  (98)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
PflFI  (163)
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  (163)
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  (172)
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.
BtgZI  (438)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its
electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
PpuMI  (491)
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  (491)
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.
XmnI  (602)
1 site
G A A N N N N T T C C T T N N N N A A G
XbaI  (625)
1 site
T C T A G A A G A T C T
FseI  (644)
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  (765)
1 site
T T A T A A A A T A T T
AanI  (765)
1 site
T T A T A A A A T A T T
PsiI  (765)
1 site
T T A T A A A A T A T T
HpaI  (785)
1 site
G T T A A C C A A T T G
MfeI  (794)
1 site
C A A T T G G T T A A C
BamHI  (887)
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  (893)
1 site
G T C G A C C A G C T G
PshAI  (958)
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.
AfeI  (1019)
1 site
A G C G C T T C G C G A
BspQI  (1027)
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  (1027)
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  (1143)
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  (1143)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NspI  (1147)
1 site
R C A T G Y Y G T A C R
BciVI  (1346)
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.
ApaLI  (1457)
1 site
G T G C A C C A C G T G
BaeGI  (1461)
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  (1461)
1 site
G K G C M C C M C G K G

Sticky ends from different Bme1580I sites may not be compatible.
RVprimer3
20-mer  /  50% GC
1 binding site
3059 .. 3078  =  20 annealed bases
Tm  =  54°C
RVprimer4
20-mer  /  65% GC
1 binding site
944 .. 963  =  20 annealed bases
Tm  =  61°C
AmpR
1992 .. 2852  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1992 .. 2783  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1992 .. 2852  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2784 .. 2852  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1992 .. 2852  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1204 .. 1792  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1204 .. 1792  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
Nluc
100 .. 615  =  516 bp
171 amino acids  =  19.1 kDa
Product: NanoLuc® luciferase
human codon-optimized
Nluc
100 .. 615  =  516 bp
171 amino acids  =  19.1 kDa
Product: NanoLuc® luciferase
human codon-optimized
SV40 poly(A) signal
664 .. 785  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
664 .. 785  =  122 bp
SV40 polyadenylation signal
pause site
3019 .. 3110  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
pause site
3019 .. 3110  =  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
2957 .. 3005  =  49 bp
synthetic polyadenylation signal
poly(A) signal
2957 .. 3005  =  49 bp
synthetic polyadenylation signal
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