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Plasmid Files

pCMV-CLuc 2

Control vector with a strong constitutive CMV promoter for expressing secreted Cypridina luciferase in mammalian cells.

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pCMV-CLuc 2 Sequence and MappCMV-CLuc 2.dna
Map and Sequence File   
Sequence Author:  New England Biolabs
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 SgrDI (6903) SspI (6786) ScaI (6462) PvuI (6352) AhdI (5982) BstZ17I (4710) BstBI (4420) RsrII (4254) BssHII (4135) MscI (3820) PstI (3791) PluTI (3741) SfoI (3739) NarI (3738) KasI (3737) SmaI (3551) BmeT110I (3550) AvaI - BsoBI - TspMI - XmaI (3549) AvrII (3528) MfeI (161) Bpu10I (180) NruI (208) MluI (228) SpeI (249) CMV enhancer NdeI (484) SnaBI (590) HindIII (889) Acc65I (895) KpnI (899) BamHI (907) BsgI (1050) AarI (1072) XcmI (1317) BstEII (1428) PshAI (1661) PmlI (1815) BmgBI (1838) EcoO109I - KflI - PpuMI (1927) Bsu36I (2479) NotI (2582) XbaI (2638) DraIII (3004) StuI (3527) pCMV-CLuc 2 6905 bp
SgrDI  (6903)
1 site
C G T C G A C G G C A G C T G C
SspI  (6786)
1 site
A A T A T T T T A T A A
ScaI  (6462)
1 site
A G T A C T T C A T G A
PvuI  (6352)
1 site
C G A T C G G C T A G C
AhdI  (5982)
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  (4710)
1 site
G T A T A C C A T A T G
BstBI  (4420)
1 site
T T C G A A A A G C T T
RsrII  (4254)
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  (4135)
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  (3820)
1 site
T G G C C A A C C G G T
PstI  (3791)
1 site
C T G C A G G A C G T C
PluTI  (3741)
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.
SfoI  (3739)
1 site
G G C G C C C C G C G G
NarI  (3738)
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.
KasI  (3737)
1 site
G G C G C C C C G C G G
SmaI  (3551)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BmeT110I  (3550)
1 site
C Y C G R G G R G C Y C
AvaI  (3549)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (3549)
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.
TspMI  (3549)
1 site
C C C G G G G G G C C C
XmaI  (3549)
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.
AvrII  (3528)
1 site
C C T A G G G G A T C C
MfeI  (161)
1 site
C A A T T G G T T A A C
Bpu10I  (180)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
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.
NruI  (208)
1 site
T C G C G A A G C G C T
MluI  (228)
1 site
A C G C G T T G C G C A
SpeI  (249)
1 site
A C T A G T T G A T C A
NdeI  (484)
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.
SnaBI  (590)
1 site
T A C G T A A T G C A T
HindIII  (889)
1 site
A A G C T T T T C G A A
Acc65I  (895)
1 site
G G T A C C C C A T G G
KpnI  (899)
1 site
G G T A C C C C A T G G
BamHI  (907)
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  (1050)
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  (1072)
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  (1317)
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  (1428)
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  (1661)
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  (1815)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BmgBI  (1838)
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  (1927)
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  (1927)
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  (1927)
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  (2479)
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  (2582)
1 site
G C G G C C G C C G C C G G C G
XbaI  (2638)
1 site
T C T A G A A G A T C T
DraIII  (3004)
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  (3527)
1 site
A G G C C T T C C G G A
CLuc
919 .. 2580  =  1662 bp
553 amino acids  =  61.5 kDa
Product: secreted Cypridina luciferase
contains 30 codon substitutions for efficient
translation in mammalian cells
CLuc
919 .. 2580  =  1662 bp
553 amino acids  =  61.5 kDa
Product: secreted Cypridina luciferase
contains 30 codon substitutions for efficient
translation in mammalian cells
AmpR
5909 .. 6769  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5909 .. 6700  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5909 .. 6769  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6701 .. 6769  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5909 .. 6769  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
3610 .. 4404  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
3610 .. 4404  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
5150 .. 5738  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5150 .. 5738  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
2771 .. 3199  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2771 .. 3199  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
3213 .. 3543  =  331 bp
SV40 enhancer and early promoter
SV40 promoter
3213 .. 3543  =  331 bp
SV40 enhancer and early promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
4578 .. 4699  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
4578 .. 4699  =  122 bp
SV40 polyadenylation signal
AmpR promoter
6770 .. 6874  =  105 bp
AmpR promoter
6770 .. 6874  =  105 bp
poly(A) signal
2589 .. 2637  =  49 bp
synthetic polyadenylation signal
poly(A) signal
2589 .. 2637  =  49 bp
synthetic polyadenylation signal
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SV40 ori
3394 .. 3529  =  136 bp
SV40 origin of replication
SV40 ori
3394 .. 3529  =  136 bp
SV40 origin of replication
Kozak sequence
913 .. 922  =  10 bp
Kozak sequence
913 .. 922  =  10 bp
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