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Plasmid Files

pCLuc-Basic 2

Mammalian promoter reporter vector encoding secreted Cypridina luciferase.

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pCLuc-Basic 2 Sequence and MappCLuc-Basic 2.dna
Map and Sequence File   
Sequence Author:  New England Biolabs
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 AatII (6060) SgrDI (6059) ZraI (6058) SspI (5942) ScaI (5618) TatI (5616) PvuI (5508) BglI (5258) BsaI (5199) AhdI (5138) BstZ17I (3866) BstBI (3576) RsrII (3410) BssHII (3291) MscI (2976) EcoRI (20) EcoRV (32) PaeR7I - PspXI - XhoI (36) MCS HindIII (45) Acc65I (51) KpnI (55) Eco53kI (59) SacI (61) BamHI (63) BsgI (206) AarI (228) XcmI (473) BstEII (584) PshAI (817) PmlI (971) BmgBI (994) EcoO109I - KflI - PpuMI (1083) Bsu36I (1635) NotI (1738) XbaI (1794) DraIII (2160) StuI (2683) AvrII (2684) TspMI - XmaI (2705) SmaI (2707) KasI (2893) NarI (2894) SfoI (2895) PluTI (2897) pCLuc-Basic 2 6061 bp
AatII  (6060)
1 site
G A C G T C C T G C A G
SgrDI  (6059)
1 site
C G T C G A C G G C A G C T G C
ZraI  (6058)
1 site
G A C G T C C T G C A G
SspI  (5942)
1 site
A A T A T T T T A T A A
ScaI  (5618)
1 site
A G T A C T T C A T G A
TatI  (5616)
1 site
W G T A C W W C A T G W
PvuI  (5508)
1 site
C G A T C G G C T A G C
BglI  (5258)
1 site
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.
BsaI  (5199)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
AhdI  (5138)
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.
BstZ17I  (3866)
1 site
G T A T A C C A T A T G
BstBI  (3576)
1 site
T T C G A A A A G C T T
RsrII  (3410)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BssHII  (3291)
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.
MscI  (2976)
1 site
T G G C C A A C C G G T
EcoRI  (20)
1 site
G A A T T C C T T A A G
EcoRV  (32)
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.
PaeR7I  (36)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (36)
1 site
V C T C G A G B B G A G C T C V
XhoI  (36)
1 site
C T C G A G G A G C T C
HindIII  (45)
1 site
A A G C T T T T C G A A
Acc65I  (51)
1 site
G G T A C C C C A T G G
KpnI  (55)
1 site
G G T A C C C C A T G G
Eco53kI  (59)
1 site
G A G C T C C T C G A G
SacI  (61)
1 site
G A G C T C C T C G A G
BamHI  (63)
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.
BsgI  (206)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
AarI  (228)
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.
XcmI  (473)
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.
BstEII  (584)
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.
PshAI  (817)
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.
PmlI  (971)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BmgBI  (994)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
EcoO109I  (1083)
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.
KflI  (1083)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
PpuMI  (1083)
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.
Bsu36I  (1635)
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.
NotI  (1738)
1 site
G C G G C C G C C G C C G G C G
XbaI  (1794)
1 site
T C T A G A A G A T C T
DraIII  (2160)
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.
StuI  (2683)
1 site
A G G C C T T C C G G A
AvrII  (2684)
1 site
C C T A G G G G A T C C
TspMI  (2705)
1 site
C C C G G G G G G C C C
XmaI  (2705)
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  (2707)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
KasI  (2893)
1 site
G G C G C C C C G C G G
NarI  (2894)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (2895)
1 site
G G C G C C C C G C G G
PluTI  (2897)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
CLuc
75 .. 1736  =  1662 bp
553 amino acids  =  61.5 kDa
Product: secreted Cypridina luciferase
contains 30 codon substitutions for efficient
translation in mammalian cells
CLuc
75 .. 1736  =  1662 bp
553 amino acids  =  61.5 kDa
Product: secreted Cypridina luciferase
contains 30 codon substitutions for efficient
translation in mammalian cells
AmpR
5065 .. 5925  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5065 .. 5856  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5065 .. 5925  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5857 .. 5925  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5065 .. 5925  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
2766 .. 3560  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
NeoR/KanR
2766 .. 3560  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
ori
4306 .. 4894  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4306 .. 4894  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
1927 .. 2355  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1927 .. 2355  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 promoter
2369 .. 2699  =  331 bp
SV40 enhancer and early promoter
SV40 promoter
2369 .. 2699  =  331 bp
SV40 enhancer and early promoter
SV40 poly(A) signal
3734 .. 3855  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3734 .. 3855  =  122 bp
SV40 polyadenylation signal
AmpR promoter
5926 .. 6030  =  105 bp
AmpR promoter
5926 .. 6030  =  105 bp
MCS
20 .. 68  =  49 bp
multiple cloning site
MCS
20 .. 68  =  49 bp
multiple cloning site
poly(A) signal
1745 .. 1793  =  49 bp
synthetic polyadenylation signal
poly(A) signal
1745 .. 1793  =  49 bp
synthetic polyadenylation signal
SV40 ori
2550 .. 2685  =  136 bp
SV40 origin of replication
SV40 ori
2550 .. 2685  =  136 bp
SV40 origin of replication
Kozak sequence
69 .. 78  =  10 bp
Kozak sequence
69 .. 78  =  10 bp
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