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Plasmid Files

pTK-CLuc

Mammalian vector with a constitutive HSV thymidine kinase promoter for expressing secreted Cypridina luciferase.

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pTK-CLuc Sequence and MappTK-CLuc.dna
Map and Sequence File   
Sequence Author:  New England Biolabs
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 ZraI (6997) SspI (6881) ScaI (6557) TatI (6555) PvuI (6447) AhdI (6077) RsrII (4349) StuI (3622) SgrDI (6998) AatII (6999) SacII (310) EcoRI (660) MluI (724) HindIII (771) Acc65I (777) KpnI (781) Eco53kI (785) SacI (787) BamHI (789) BsgI (932) AarI (954) XcmI (1199) BstEII (1310) PshAI (1543) PmlI (1697) BmgBI (1720) KflI (1809) Bsu36I (2361) NotI (2464) AgeI (2471) PaeR7I - XhoI (2477) XbaI (2489) HpaI (2631) MfeI (2640) BspDI - ClaI (2726) DraIII (3099) pTK-CLuc 7000 bp
ZraI  (6997)
1 site
G A C G T C C T G C A G
SspI  (6881)
1 site
A A T A T T T T A T A A
ScaI  (6557)
1 site
A G T A C T T C A T G A
TatI  (6555)
1 site
W G T A C W W C A T G W
PvuI  (6447)
1 site
C G A T C G G C T A G C
AhdI  (6077)
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.
RsrII  (4349)
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.
StuI  (3622)
1 site
A G G C C T T C C G G A
SgrDI  (6998)
1 site
C G T C G A C G G C A G C T G C
AatII  (6999)
1 site
G A C G T C C T G C A G
SacII  (310)
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.
EcoRI  (660)
1 site
G A A T T C C T T A A G
MluI  (724)
1 site
A C G C G T T G C G C A
HindIII  (771)
1 site
A A G C T T T T C G A A
Acc65I  (777)
1 site
G G T A C C C C A T G G
KpnI  (781)
1 site
G G T A C C C C A T G G
Eco53kI  (785)
1 site
G A G C T C C T C G A G
SacI  (787)
1 site
G A G C T C C T C G A G
BamHI  (789)
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  (932)
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  (954)
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  (1199)
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  (1310)
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  (1543)
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  (1697)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BmgBI  (1720)
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.
KflI  (1809)
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.
Bsu36I  (2361)
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  (2464)
1 site
G C G G C C G C C G C C G G C G
AgeI  (2471)
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.
PaeR7I  (2477)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (2477)
1 site
C T C G A G G A G C T C
XbaI  (2489)
1 site
T C T A G A A G A T C T
HpaI  (2631)
1 site
G T T A A C C A A T T G
MfeI  (2640)
1 site
C A A T T G G T T A A C
BspDI  (2726)
1 site
A T C G A T T A G C T A
ClaI  (2726)
1 site
A T C G A T T A G C T A
DraIII  (3099)
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.
CLuc
801 .. 2462  =  1662 bp
553 amino acids  =  61.5 kDa
Product: secreted Cypridina luciferase
contains 30 codon substitutions for efficient
translation in mammalian cells
CLuc
801 .. 2462  =  1662 bp
553 amino acids  =  61.5 kDa
Product: secreted Cypridina luciferase
contains 30 codon substitutions for efficient
translation in mammalian cells
AmpR
6004 .. 6864  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6004 .. 6795  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6004 .. 6864  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6796 .. 6864  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6004 .. 6864  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
3705 .. 4499  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
3705 .. 4499  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
HSV TK promoter
19 .. 770  =  752 bp
herpes simplex virus thymidine kinase promoter
HSV TK promoter
19 .. 770  =  752 bp
herpes simplex virus thymidine kinase promoter
ori
5245 .. 5833  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5245 .. 5833  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
2866 .. 3294  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2866 .. 3294  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 promoter
3308 .. 3638  =  331 bp
SV40 enhancer and early promoter
SV40 promoter
3308 .. 3638  =  331 bp
SV40 enhancer and early promoter
SV40 poly(A) signal
2510 .. 2631  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2510 .. 2631  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
4673 .. 4794  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
4673 .. 4794  =  122 bp
SV40 polyadenylation signal
AmpR promoter
6865 .. 6969  =  105 bp
AmpR promoter
6865 .. 6969  =  105 bp
MCS
2463 .. 2494  =  32 bp
multiple cloning site
MCS
2463 .. 2494  =  32 bp
multiple cloning site
SV40 ori
3489 .. 3624  =  136 bp
SV40 origin of replication
SV40 ori
3489 .. 3624  =  136 bp
SV40 origin of replication
Kozak sequence
795 .. 804  =  10 bp
Kozak sequence
795 .. 804  =  10 bp
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