pNL1.1.CMV[Nluc CMV]

Co-transfection vector for expressing NanoLuc® luciferase under control of the strong CMV promoter.

Sequence Author: Promega

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AarI - BfuAI - BspMI (3831) RVprimer3 (3810 .. 3829) BsmBI (3658) BstZ17I (3326) SacII (3210) PvuI (3186) Bsu36I (3172) AhdI (2816) BstEII (2741) BstXI - PstI (2738) AleI (2736) NotI (2714) BspHI (2614) AlwNI (2310) BaeGI - Bme1580I (2212) ApaLI (2208) BciVI (2097) BssSI (2067) NspI (1898) SfiI (8) BsrGI (109) AseI (173) NdeI (400) BsaAI - SnaBI (506) Eco53kI (732) BanII - SacI (734) SfiI (818) PflFI - Tth111I (922) EcoNI (931) AvaI - BmeT110I - BsoBI (964) Bpu10I (1000) PpuMI - SanDI (1250) XmnI (1361) XbaI (1376) FseI (1395) PsiI (1516) HpaI (1536) MfeI (1545) BamHI (1638) SalI (1644) PshAI (1709) RVprimer4 (1695 .. 1714) AfeI (1770) BspQI - SapI (1778) AflIII - PciI (1894) pNL1.1.CMV[Nluc/CMV] 3861 bp
AarI  (3831)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Cleavage may be enhanced when more than one copy of the AarI recognition sequence is present.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its electrophoretic mobility.
BfuAI  (3831)
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  (3831)
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  (3658)
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.
BstZ17I  (3326)
1 site
G T A T A C C A T A T G
SacII  (3210)
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  (3186)
1 site
C G A T C G G C T A G C
Bsu36I  (3172)
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.
AhdI  (2816)
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  (2741)
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  (2738)
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  (2738)
1 site
C T G C A G G A C G T C
AleI  (2736)
1 site
C A C N N N N G T G G T G N N N N C A C
NotI  (2714)
1 site
G C G G C C G C C G C C G G C G
BspHI  (2614)
1 site
T C A T G A A G T A C T
AlwNI  (2310)
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.
BaeGI  (2212)
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  (2212)
1 site
G K G C M C C M C G K G

Sticky ends from different Bme1580I sites may not be compatible.
ApaLI  (2208)
1 site
G T G C A C C A C G T G
BciVI  (2097)
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  (2067)
1 site
C A C G A G G T G C T C
NspI  (1898)
1 site
R C A T G Y Y G T A C R
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.
BsrGI  (109)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
AseI  (173)
1 site
A T T A A T T A A T T A
NdeI  (400)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional nucleotides.
BsaAI  (506)
1 site
Y A C G T R R T G C A Y
SnaBI  (506)
1 site
T A C G T A A T G C A T
Eco53kI  (732)
1 site
G A G C T C C T C G A G
BanII  (734)
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  (734)
1 site
G A G C T C C T C G A G
SfiI  (818)
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.
PflFI  (922)
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  (922)
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  (931)
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.
AvaI  (964)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BmeT110I  (964)
1 site
C Y C G R G G R G C Y C
BsoBI  (964)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
Bpu10I  (1000)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
PpuMI  (1250)
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  (1250)
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  (1361)
1 site
G A A N N N N T T C C T T N N N N A A G
XbaI  (1376)
1 site
T C T A G A A G A T C T
FseI  (1395)
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  (1516)
1 site
T T A T A A A A T A T T
HpaI  (1536)
1 site
G T T A A C C A A T T G
MfeI  (1545)
1 site
C A A T T G G T T A A C
BamHI  (1638)
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  (1644)
1 site
G T C G A C C A G C T G
PshAI  (1709)
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  (1770)
1 site
A G C G C T T C G C G A
BspQI  (1778)
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  (1778)
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  (1894)
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  (1894)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
RVprimer3
20-mer  /  50% GC
1 binding site
3810 .. 3829  =  20 annealed bases
Tm  =  54°C
RVprimer4
20-mer  /  65% GC
1 binding site
1695 .. 1714  =  20 annealed bases
Tm  =  62°C
AmpR
2743 .. 3603  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   2743 .. 3534  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2743 .. 3603  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3535 .. 3603  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2743 .. 3603  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1955 .. 2543  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1955 .. 2543  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
Nluc
859 .. 1374  =  516 bp
171 amino acids  =  19.1 kDa
Product: NanoLuc® luciferase
human codon-optimized
Nluc
859 .. 1374  =  516 bp
171 amino acids  =  19.1 kDa
Product: NanoLuc® luciferase
human codon-optimized
CMV enhancer
227 .. 530  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
227 .. 530  =  304 bp
human cytomegalovirus immediate early enhancer
CMV promoter
531 .. 734  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
531 .. 734  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 poly(A) signal
1415 .. 1536  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1415 .. 1536  =  122 bp
SV40 polyadenylation signal
pause site
3770 .. 3861  =  92 bp
RNA polymerase II transcriptional pause signal from the human α2 globin gene
pause site
3770 .. 3861  =  92 bp
RNA polymerase II transcriptional pause signal from the human α2 globin gene
poly(A) signal
3708 .. 3756  =  49 bp
synthetic polyadenylation signal
poly(A) signal
3708 .. 3756  =  49 bp
synthetic polyadenylation signal
ORF:  859 .. 1374  =  516 bp
ORF:  171 amino acids  =  19.1 kDa
ORF:  2873 .. 3139  =  267 bp
ORF:  88 amino acids  =  9.3 kDa
ORF:  805 .. 1122  =  318 bp
ORF:  105 amino acids  =  11.7 kDa
ORF:  1501 .. 1743  =  243 bp
ORF:  80 amino acids  =  9.0 kDa
ORF:  2743 .. 3603  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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