pMetLuc2-Reporter

Mammalian vector for measuring the activity of promoter and enhancer sequences using secreted Metridia luciferase.

Sequence Author: Clontech (TaKaRa)

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AfeI (14) ScaI (4096) poly(A) signal PfoI (2889) RsrII (2630) BsrDI (2347) PflFI - Tth111I (2232) FspI (2216) BglII (27) PaeR7I - XhoI (31) Eco53kI (36) SacI (38) HindIII (40) EcoRI (47) PstI (56) SalI (57) AccI (58) Acc65I (63) KpnI (67) SacII (70) PspOMI (71) ApaI (75) BamHI (78) AgeI (84) AleI (121) EcoNI (139) BclI * (257) XcmI (297) Bpu10I (424) BsrGI (458) NotI (758) XbaI * (768) MfeI (864) HpaI (877) BtsI - BtsĪ±I (953) AflII (996) DraIII (1230) CsiI - SexAI * (1703) SV40 promoter SfiI (1889) BseRI (1932) StuI (1935) pMetLuc2-Reporter 4250 bp
AfeI  (14)
1 site
A G C G C T T C G C G A
ScaI  (4096)
1 site
A G T A C T T C A T G A
PfoI  (2889)
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  (2630)
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  (2347)
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  (2232)
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  (2232)
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  (2216)
1 site
T G C G C A A C G C G T
BglII  (27)
1 site
A G A T C T T C T A 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
Eco53kI  (36)
1 site
G A G C T C C T C G A G
SacI  (38)
1 site
G A G C T C C T C G A G
HindIII  (40)
1 site
A A G C T T T T C G A A
EcoRI  (47)
1 site
G A A T T C C T T A A G
PstI  (56)
1 site
C T G C A G G A C G T C
SalI  (57)
1 site
G T C G A C C A G C T G
AccI  (58)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ā‰„13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
Acc65I  (63)
1 site
G G T A C C C C A T G G
KpnI  (67)
1 site
G G T A C C C C A T G G
SacII  (70)
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  (71)
1 site
G G G C C C C C C G G G
ApaI  (75)
1 site
G G G C C C C C C G G G

ApaI can be used between 25Ā°C and 37Ā°C.
BamHI  (78)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HFĀ® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
AgeI  (84)
1 site
A C C G G T T G G C C A
AleI  (121)
1 site
C A C N N N N G T G G T G N N N N C A C
EcoNI  (139)
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  (257)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55Ā°C, but is 50% active at 37Ā°C.
XcmI  (297)
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  (424)
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.
BsrGI  (458)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37Ā°C, but is even more active at 60Ā°C.
NotI  (758)
1 site
G C G G C C G C C G C C G G C G
XbaI  (768)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
MfeI  (864)
1 site
C A A T T G G T T A A C
HpaI  (877)
1 site
G T T A A C C A A T T G
BtsI  (953)
1 site
G C A G T G N N C G T C A C
BtsĪ±I  (953)
1 site
G C A G T G N N C G T C A C

Sticky ends from different BtsĪ±I sites may not be compatible.
AflII  (996)
1 site
C T T A A G G A A T T C
DraIII  (1230)
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.
CsiI  (1703)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
SexAI  (1703)
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  (1889)
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  (1932)
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 37Ā°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
StuI  (1935)
1 site
A G G C C T T C C G G A
NeoR/KanR
1986 .. 2780  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (GeneticinĀ®)
NeoR/KanR
1986 .. 2780  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (GeneticinĀ®)
MetLuc
97 .. 756  =  660 bp
219 amino acids  =  23.9 kDa
2 segments
   Segment 1:  signal sequence  
   97 .. 147  =  51 bp
   17 amino acids  =  1.9 kDa
Product: secreted Metridia luciferase
human codon-optimized
MetLuc
97 .. 756  =  660 bp
219 amino acids  =  23.9 kDa
2 segments
   Segment 2:  
   148 .. 756  =  609 bp
   202 amino acids  =  22.0 kDa
Product: secreted Metridia luciferase
human codon-optimized
MetLuc
97 .. 756  =  660 bp
219 amino acids  =  23.9 kDa
2 segments
Product: secreted Metridia luciferase
human codon-optimized
ori
3388 .. 3976  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3388 .. 3976  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
1006 .. 1461  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
1006 .. 1461  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
SV40 promoter
1594 .. 1951  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
1594 .. 1951  =  358 bp
SV40 enhancer and early promoter
SV40 poly(A) signal
878 .. 999  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
878 .. 999  =  122 bp
SV40 polyadenylation signal
AmpR promoter
1488 .. 1592  =  105 bp
AmpR promoter
1488 .. 1592  =  105 bp
pause site
4100 .. 4191  =  92 bp
RNA polymerase II transcriptional pause signal from the human Ī±2 globin gene
pause site
4100 .. 4191  =  92 bp
RNA polymerase II transcriptional pause signal from the human Ī±2 globin gene
MCS
12 .. 89  =  78 bp
multiple cloning site
MCS
12 .. 89  =  78 bp
multiple cloning site
poly(A) signal
4038 .. 4086  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4038 .. 4086  =  49 bp
synthetic polyadenylation signal
HSV TK poly(A) signal
3012 .. 3059  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
3012 .. 3059  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
SV40 ori
1802 .. 1937  =  136 bp
SV40 origin of replication
SV40 ori
1802 .. 1937  =  136 bp
SV40 origin of replication
ORF:  97 .. 756  =  660 bp
ORF:  219 amino acids  =  23.9 kDa
ORF:  2158 .. 2544  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  2801 .. 3250  =  450 bp
ORF:  149 amino acids  =  16.3 kDa
ORF:  1986 .. 2780  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  4190 .. 464  =  525 bp
ORF:  174 amino acids
ORF:  483 .. 740  =  258 bp
ORF:  85 amino acids  =  9.4 kDa
ORF:  2295 .. 2831  =  537 bp
ORF:  178 amino acids  =  19.8 kDa
ORF:  3006 .. 3239  =  234 bp
ORF:  77 amino acids  =  8.6 kDa
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