pSF-CMV-RSV-RLuc AscI

Mammalian vector for co-expressing a gene together with Renilla luciferase expressed from the Rous sarcoma virus promoter.

Sequence Author: Oxford Genetics

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BglII (232) AsiSI (5) PmeI (5610) RsrII (5303) PflFI - Tth111I (4905) RBS PmeI (4562) PpuMI - SanDI (4496) AscI (4405) AanI (3677) BstAPI (3573) AleI (3361) SphI (3202) Bsu36I (3070) NruI (2978) AscI (2921) FseI (2775) NaeI (2773) SnaBI (566) CMV promoter BglII (816) EagI - NotI (858) HindIII (869) Eco53kI (879) SacI (881) EcoRI (885) MCS KpnI (901) NcoI (905) KpnI (915) EcoRV (921) AbsI - PaeR7I - PspXI - XhoI (928) XbaI (937) BseRI - BsgI (940) stop codons BspDI - ClaI (977) BamHI (986) StuI (996) NheI (1002) BmtI (1006) stop codons SV40 poly(A) signal AanI (1152) HpaI (1172) T7 terminator PstI - SbfI (1518) 3' β-globin insulator BstEII (1624) PacI (1650) SwaI (1776) BssSI (1953) AlwNI (2196) BspHI (2500) SwaI (2669) NgoMIV (2771) pSF-CMV-RSV- RLuc AscI 5729 bp
BglII  (232)
2 sites
A G A T C T T C T A G A
AsiSI  (5)
1 site
G C G A T C G C C G C T A G C G
PmeI  (5610)
2 sites
G T T T A A A C C A A A T T T G
RsrII  (5303)
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  (4905)
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  (4905)
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  (4562)
2 sites
G T T T A A A C C A A A T T T G
PpuMI  (4496)
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  (4496)
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  (4405)
2 sites
G G C G C G C C C C G C G C G G
AanI  (3677)
2 sites
T T A T A A A A T A T T
BstAPI  (3573)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
AleI  (3361)
1 site
C A C N N N N G T G G T G N N N N C A C
SphI  (3202)
1 site
G C A T G C C G T A C G
Bsu36I  (3070)
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.
NruI  (2978)
1 site
T C G C G A A G C G C T
AscI  (2921)
2 sites
G G C G C G C C C C G C G C G G
FseI  (2775)
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.
NaeI  (2773)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI recognition sequence.
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
HindIII  (869)
1 site
A A G C T T T T C G A A
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
KpnI  (901)
2 sites
G G T A C C C C A T G G
NcoI  (905)
1 site
C C A T G G G G T A C C
KpnI  (915)
2 sites
G G T A C C C C A T G G
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
BamHI  (986)
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.
StuI  (996)
1 site
A G G C C T T C C G G A
NheI  (1002)
1 site
G C T A G C C G A T C G
BmtI  (1006)
1 site
G C T A G C C G A T C G
AanI  (1152)
2 sites
T T A T A A A A T A T T
HpaI  (1172)
1 site
G T T A A C C A A T T G
PstI  (1518)
1 site
C T G C A G G A C G T C
SbfI  (1518)
1 site
C C T G C A G G G G A C G T C C
BstEII  (1624)
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.
PacI  (1650)
1 site
T T A A T T A A A A T T A A T T
SwaI  (1776)
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.
BssSI  (1953)
1 site
C A C G A G G T G C T C
AlwNI  (2196)
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.
BspHI  (2500)
1 site
T C A T G A A G T A C T
SwaI  (2669)
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.
NgoMIV  (2771)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
RLuc
3385 .. 4323  =  939 bp
312 amino acids  =  36.1 kDa
Product: Renilla luciferase
codon-optimized synthetic gene
RLuc
3385 .. 4323  =  939 bp
312 amino acids  =  36.1 kDa
Product: Renilla luciferase
codon-optimized synthetic gene
NeoR/KanR
4659 .. 5453  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
4659 .. 5453  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
ori
1841 .. 2429  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1841 .. 2429  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CMV enhancer
287 .. 590  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
287 .. 590  =  304 bp
human cytomegalovirus immediate early enhancer
RSV promoter
3110 .. 3372  =  263 bp
Rous sarcoma virus enhancer/promoter
RSV promoter
3110 .. 3372  =  263 bp
Rous sarcoma virus enhancer/promoter
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
1363 .. 1499  =  137 bp
transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)
rrnG terminator
1363 .. 1499  =  137 bp
transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)
rrnG terminator
5466 .. 5602  =  137 bp
transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)
rrnG terminator
5466 .. 5602  =  137 bp
transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)
SV40 poly(A) signal
1051 .. 1172  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1051 .. 1172  =  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
1544 .. 1615  =  72 bp
insulator downstream of the human β-globin locus (Farrell et al., 2002)
3' β-globin insulator
1544 .. 1615  =  72 bp
insulator downstream of the human β-globin locus (Farrell et al., 2002)
poly(A) signal
4345 .. 4393  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4345 .. 4393  =  49 bp
synthetic polyadenylation signal
T7 terminator
1287 .. 1333  =  47 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
1287 .. 1333  =  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
1009 .. 1019  =  11 bp
stop codons in all three reading frames
stop codons
1009 .. 1019  =  11 bp
stop codons in all three reading frames
RBS
4646 .. 4651  =  6 bp
Shine-Dalgarno ribosome binding site
RBS
4646 .. 4651  =  6 bp
Shine-Dalgarno ribosome binding site
ORF:  3385 .. 4323  =  939 bp
ORF:  312 amino acids  =  36.1 kDa
ORF:  4831 .. 5217  =  387 bp
ORF:  128 amino acids  =  14.7 kDa
ORF:  5491 .. 196  =  435 bp
ORF:  144 amino acids  =  16.1 kDa
ORF:  1388 .. 1819  =  432 bp
ORF:  143 amino acids  =  16.3 kDa
ORF:  4659 .. 5453  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  2878 .. 3267  =  390 bp
ORF:  129 amino acids  =  14.3 kDa
ORF:  3313 .. 4239  =  927 bp
ORF:  308 amino acids  =  36.5 kDa
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