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Plasmid Files

pCRE-MetLuc2-Reporter

Vector for monitoring cAMP-mediated signal transduction using secreted Metridia luciferase.

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pCRE-MetLuc2-Reporter.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Clontech (TaKaRa)
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AfeI (14) ScaI (4335) PfoI (3128) RsrII (2869) BsrDI (2586) PflFI - Tth111I (2471) FspI (2455) StuI (2174) PaeR7I - XhoI (31) HindIII (279) PstI (295) SalI (296) AccI (297) Acc65I (302) KpnI (306) SacII (309) PspOMI (310) ApaI (314) BamHI (317) AgeI (323) AleI (360) EcoNI (378) BclI * (496) XcmI (536) Bpu10I (663) BsrGI (697) NotI (997) XbaI * (1007) MfeI (1103) HpaI (1116) Bts α I (1192) AflII (1235) DraIII (1469) SexAI * (1942) SfiI (2128) BseRI (2171) pCRE-MetLuc2-Reporter 4489 bp
AfeI  (14)
1 site
A G C G C T T C G C G A
ScaI  (4335)
1 site
A G T A C T T C A T G A
PfoI  (3128)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
RsrII  (2869)
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.
BsrDI  (2586)
1 site
G C A A T G N N C G T T A C

Sticky ends from different BsrDI sites may not be compatible.
PflFI  (2471)
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  (2471)
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.
FspI  (2455)
1 site
T G C G C A A C G C G T
StuI  (2174)
1 site
A G G C C T T C C G G A
PaeR7I  (31)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (31)
1 site
C T C G A G G A G C T C
HindIII  (279)
1 site
A A G C T T T T C G A A
PstI  (295)
1 site
C T G C A G G A C G T C
SalI  (296)
1 site
G T C G A C C A G C T G
AccI  (297)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
Acc65I  (302)
1 site
G G T A C C C C A T G G
KpnI  (306)
1 site
G G T A C C C C A T G G
SacII  (309)
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.
PspOMI  (310)
1 site
G G G C C C C C C G G G
ApaI  (314)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BamHI  (317)
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.
AgeI  (323)
1 site
A C C G G T T G G C C A
AleI  (360)
1 site
C A C N N N N G T G G T G N N N N C A C
EcoNI  (378)
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.
BclI  (496)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
XcmI  (536)
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.
Bpu10I  (663)
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.
BsrGI  (697)
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.
NotI  (997)
1 site
G C G G C C G C C G C C G G C G
XbaI  (1007)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
MfeI  (1103)
1 site
C A A T T G G T T A A C
HpaI  (1116)
1 site
G T T A A C C A A T T G
BtsαI  (1192)
1 site
G C A G T G N N C G T C A C

Sticky ends from different BtsαI sites may not be compatible.
AflII  (1235)
1 site
C T T A A G G A A T T C
DraIII  (1469)
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.
SexAI  (1942)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
SfiI  (2128)
1 site
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.
BseRI  (2171)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
NeoR/KanR
2225 .. 3019  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
2225 .. 3019  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
MetLuc
336 .. 995  =  660 bp
219 amino acids  =  23.9 kDa
   Segment 1:  signal sequence  
   336 .. 386  =  51 bp
   17 amino acids  =  1.9 kDa
Product: secreted Metridia luciferase
human codon-optimized
MetLuc
336 .. 995  =  660 bp
219 amino acids  =  23.9 kDa
   Segment 2:  
   387 .. 995  =  609 bp
   202 amino acids  =  22.0 kDa
Product: secreted Metridia luciferase
human codon-optimized
MetLuc
336 .. 995  =  660 bp
219 amino acids  =  23.9 kDa
2 segments
Product: secreted Metridia luciferase
human codon-optimized
ori
3627 .. 4215  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3627 .. 4215  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
1245 .. 1700  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
1245 .. 1700  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
SV40 promoter
1833 .. 2190  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
1833 .. 2190  =  358 bp
SV40 enhancer and early promoter
TATA-like promoter region
130 .. 278  =  149 bp
TATA-like promoter region from the herpes simplex virus thymidine kinase promoter
TATA-like promoter region
130 .. 278  =  149 bp
TATA-like promoter region from the herpes simplex virus thymidine kinase promoter
SV40 poly(A) signal
1117 .. 1238  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1117 .. 1238  =  122 bp
SV40 polyadenylation signal
AmpR promoter
1727 .. 1831  =  105 bp
AmpR promoter
1727 .. 1831  =  105 bp
pause site
4339 .. 4430  =  92 bp
RNA polymerase II transcriptional pause signal from the human α2 globin gene
pause site
4339 .. 4430  =  92 bp
RNA polymerase II transcriptional pause signal from the human α2 globin gene
CRE
48 .. 113  =  66 bp
cyclic AMP response element (3 copies)
CRE
48 .. 113  =  66 bp
cyclic AMP response element (3 copies)
poly(A) signal
4277 .. 4325  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4277 .. 4325  =  49 bp
synthetic polyadenylation signal
HSV TK poly(A) signal
3251 .. 3298  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
3251 .. 3298  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
SV40 ori
2041 .. 2176  =  136 bp
SV40 origin of replication
SV40 ori
2041 .. 2176  =  136 bp
SV40 origin of replication
ORF:  3040 .. 3489  =  450 bp
ORF:  149 amino acids  =  16.3 kDa
ORF:  212 .. 1018  =  807 bp
ORF:  268 amino acids  =  30.5 kDa
ORF:  2225 .. 3019  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  114 .. 995  =  882 bp
ORF:  293 amino acids  =  32.0 kDa
ORF:  2397 .. 2783  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  53 .. 703  =  651 bp
ORF:  216 amino acids  =  23.3 kDa
ORF:  722 .. 979  =  258 bp
ORF:  85 amino acids  =  9.4 kDa
ORF:  2534 .. 3070  =  537 bp
ORF:  178 amino acids  =  19.8 kDa
ORF:  3245 .. 3478  =  234 bp
ORF:  77 amino acids  =  8.6 kDa
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