pSF-CMV-EMCV-FLuc

Mammalian vector for co-expressing a gene together with firefly luciferase expressed from the encephalomyocarditis virus IRES.

Sequence Author: Oxford Genetics

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PmeI (6342) RsrII (6035) PflFI - Tth111I (5637) PmeI (5294) PpuMI - SanDI (5228) AscI - BssHII (5137) FseI (4991) SwaI (4885) AlwNI (4412) SwaI (3992) PacI (3866) BstEII (3840) 3' β-globin insulator PstI - SbfI (3734) MfeI (3397) AanI - PsiI (3368) BtsI (3314) stop codons AsiSI - PvuI (5) BglII (232) CMV enhancer SnaBI (566) BglII (816) EagI - NotI (858) Eco53kI (879) SacI (881) EcoRI (885) NcoI (905) EcoRV (921) AbsI - PaeR7I - PspXI - XhoI (928) XbaI (937) BseRI - BsgI (940) stop codons BspDI - ClaI (977) PspOMI (1101) ApaI (1105) AvrII (1139) PmlI (1304) AarI (1327) PflMI (1441) BmgBI (1530) PvuII (1639) ScaI (1730) BsrGI (2055) AleI (2063) FspI (2387) KasI (2512) NarI (2513) SfoI (2514) PluTI (2516) BlpI (2519) AgeI (2696) SexAI * (2904) BpmI (2945) NheI (3218) BmtI (3222) pSF-CMV-EMCV-FLuc 6461 bp
PmeI  (6342)
2 sites
G T T T A A A C C A A A T T T G
RsrII  (6035)
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.
PflFI  (5637)
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  (5637)
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.
PmeI  (5294)
2 sites
G T T T A A A C C A A A T T T G
PpuMI  (5228)
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.
SanDI  (5228)
1 site
G G G W C C C C C C W G G G

Sticky ends from different SanDI sites may not be compatible.
AscI  (5137)
1 site
G G C G C G C C C C G C G C G G
BssHII  (5137)
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.
FseI  (4991)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
SwaI  (4885)
2 sites
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
AlwNI  (4412)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
SwaI  (3992)
2 sites
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
PacI  (3866)
1 site
T T A A T T A A A A T T A A T T
BstEII  (3840)
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.
PstI  (3734)
1 site
C T G C A G G A C G T C
SbfI  (3734)
1 site
C C T G C A G G G G A C G T C C
MfeI  (3397)
1 site
C A A T T G G T T A A C
AanI  (3368)
1 site
T T A T A A A A T A T T
PsiI  (3368)
1 site
T T A T A A A A T A T T
BtsI  (3314)
1 site
G C A G T G N N C G T C A C
AsiSI  (5)
1 site
G C G A T C G C C G C T A G C G
PvuI  (5)
1 site
C G A T C G G C T A G C
BglII  (232)
2 sites
A G A T C T T C T A G A
SnaBI  (566)
1 site
T A C G T A A T G C A T
BglII  (816)
2 sites
A G A T C T T C T A G A
EagI  (858)
1 site
C G G C C G G C C G G C
NotI  (858)
1 site
G C G G C C G C C G C C G G C G
Eco53kI  (879)
1 site
G A G C T C C T C G A G
SacI  (881)
1 site
G A G C T C C T C G A G
EcoRI  (885)
1 site
G A A T T C C T T A A G
NcoI  (905)
1 site
C C A T G G G G T A C C
EcoRV  (921)
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.
AbsI  (928)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (928)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (928)
1 site
V C T C G A G B B G A G C T C V
XhoI  (928)
1 site
C T C G A G G A G C T C
XbaI  (937)
1 site
T C T A G A A G A T C T
BseRI  (940)
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.
BsgI  (940)
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).
BspDI  (977)
1 site
A T C G A T T A G C T A
ClaI  (977)
1 site
A T C G A T T A G C T A
PspOMI  (1101)
1 site
G G G C C C C C C G G G
ApaI  (1105)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
AvrII  (1139)
1 site
C C T A G G G G A T C C
PmlI  (1304)
1 site
C A C G T G G T G C A C
AarI  (1327)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Cleavage may be enhanced when more than one copy of the AarI recognition sequence is present.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its electrophoretic mobility.
PflMI  (1441)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
BmgBI  (1530)
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.
PvuII  (1639)
1 site
C A G C T G G T C G A C
ScaI  (1730)
1 site
A G T A C T T C A T G A
BsrGI  (2055)
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.
AleI  (2063)
1 site
C A C N N N N G T G G T G N N N N C A C
FspI  (2387)
1 site
T G C G C A A C G C G T
KasI  (2512)
1 site
G G C G C C C C G C G G
NarI  (2513)
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  (2514)
1 site
G G C G C C C C G C G G
PluTI  (2516)
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.
BlpI  (2519)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
AgeI  (2696)
1 site
A C C G G T T G G C C A
SexAI  (2904)
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.
BpmI  (2945)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
NheI  (3218)
1 site
G C T A G C C G A T C G
BmtI  (3222)
1 site
G C T A G C C G A T C G
luciferase
1565 .. 3217  =  1653 bp
550 amino acids  =  60.6 kDa
Product: firefly luciferase
synthetic luc2 version of the luciferase gene
luciferase
1565 .. 3217  =  1653 bp
550 amino acids  =  60.6 kDa
Product: firefly luciferase
synthetic luc2 version of the luciferase gene
NeoR/KanR
5391 .. 6185  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
5391 .. 6185  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
ori
4057 .. 4645  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4057 .. 4645  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
IRES
1012 .. 1562  =  551 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES
1012 .. 1562  =  551 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
CMV enhancer
287 .. 590  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
287 .. 590  =  304 bp
human cytomegalovirus immediate early enhancer
CMV promoter
591 .. 794  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
591 .. 794  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
rrnG terminator
3579 .. 3715  =  137 bp
transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)
rrnG terminator
3579 .. 3715  =  137 bp
transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)
rrnG terminator
6198 .. 6334  =  137 bp
transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)
rrnG terminator
6198 .. 6334  =  137 bp
transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)
SV40 poly(A) signal
3267 .. 3388  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3267 .. 3388  =  122 bp
SV40 polyadenylation signal
MCS
857 .. 942  =  86 bp
multiple cloning site
MCS
857 .. 942  =  86 bp
multiple cloning site
5' β-globin insulator
18 .. 89  =  72 bp
insulator upstream of the human β-globin locus (Farrell et al., 2002)
5' β-globin insulator
18 .. 89  =  72 bp
insulator upstream of the human β-globin locus (Farrell et al., 2002)
3' β-globin insulator
3760 .. 3831  =  72 bp
insulator downstream of the human β-globin locus (Farrell et al., 2002)
3' β-globin insulator
3760 .. 3831  =  72 bp
insulator downstream of the human β-globin locus (Farrell et al., 2002)
T7 terminator
3503 .. 3549  =  47 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
3503 .. 3549  =  47 bp
transcription terminator for bacteriophage T7 RNA polymerase
stop codons
961 .. 971  =  11 bp
stop codons in all three reading frames
stop codons
961 .. 971  =  11 bp
stop codons in all three reading frames
stop codons
3225 .. 3235  =  11 bp
stop codons in all three reading frames
stop codons
3225 .. 3235  =  11 bp
stop codons in all three reading frames
RBS
5378 .. 5383  =  6 bp
Shine-Dalgarno ribosome binding site
RBS
5378 .. 5383  =  6 bp
Shine-Dalgarno ribosome binding site
Kozak sequence
903 .. 909  =  7 bp
Kozak sequence
903 .. 909  =  7 bp
RBS
893 .. 898  =  6 bp
Shine-Dalgarno ribosome binding site
RBS
893 .. 898  =  6 bp
Shine-Dalgarno ribosome binding site
ATG
907 .. 909  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
907 .. 909  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  3604 .. 4035  =  432 bp
ORF:  143 amino acids  =  16.3 kDa
ORF:  5563 .. 5949  =  387 bp
ORF:  128 amino acids  =  14.7 kDa
ORF:  6223 .. 196  =  435 bp
ORF:  144 amino acids  =  16.1 kDa
ORF:  1565 .. 3217  =  1653 bp
ORF:  550 amino acids  =  60.6 kDa
ORF:  5391 .. 6185  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  1496 .. 1792  =  297 bp
ORF:  98 amino acids  =  10.7 kDa
ORF:  2561 .. 2821  =  261 bp
ORF:  86 amino acids  =  8.9 kDa
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