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Plasmid Files

pSF-pA-CMVe-FLuc

Vector encoding the CMV enhancer and firefly luciferase, for low-background measurements of promoter activity.

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pSF-pA-CMVe-FLuc Sequence and MappSF-pA-CMVe-FLuc.dna
Map and Sequence File   
Sequence Author:  Oxford Genetics
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 PmeI (5854) RsrII (5547) BsrDI (5264) PflFI - Tth111I (5149) PmeI (4806) PpuMI - SanDI (4740) AscI - BssHII (4649) FseI (4503) SwaI (4397) AlwNI (3924) ApaLI (3822) SwaI (3504) PacI (3378) BstEII (3352) 3' β- globin insulator PstI - SbfI (3246) AsiSI - PvuI (5) BglII (232) AseI (244) CMV enhancer SnaBI (577) NruI (710) PshAI (716) poly(A) signal AarI (854) SalI (885) AccI (886) SpeI (893) TspMI - XmaI (901) SmaI (903) PspOMI (909) ApaI (913) BstBI (918) BglII (926) EagI - NotI (968) HindIII (979) Eco53kI (989) SacI (991) EcoRI (995) Acc65I (1007) KpnI (1011) NcoI (1015) BbsI (1075) PvuII (1091) BsrGI (1507) AleI (1515) FspI (1839) KasI (1964) NarI (1965) SfoI (1966) PluTI (1968) BlpI (1971) AgeI (2148) DraIII (2163) SexAI * (2356) BpmI (2397) XbaI (2665) BseRI - BsgI (2668) stop codons BspDI - ClaI (2705) BamHI (2714) StuI (2724) NheI (2730) BmtI (2734) stop codons AanI - PsiI (2880) MfeI (2909) pSF-pA-CMVe-FLuc 5973 bp
PmeI  (5854)
2 sites
G T T T A A A C C A A A T T T G
RsrII  (5547)
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  (5264)
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  (5149)
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  (5149)
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  (4806)
2 sites
G T T T A A A C C A A A T T T G
PpuMI  (4740)
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  (4740)
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  (4649)
1 site
G G C G C G C C C C G C G C G G
BssHII  (4649)
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  (4503)
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  (4397)
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  (3924)
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.
ApaLI  (3822)
1 site
G T G C A C C A C G T G
SwaI  (3504)
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  (3378)
1 site
T T A A T T A A A A T T A A T T
BstEII  (3352)
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  (3246)
1 site
C T G C A G G A C G T C
SbfI  (3246)
1 site
C C T G C A G G G G A C G T C 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
AseI  (244)
1 site
A T T A A T T A A T T A
SnaBI  (577)
1 site
T A C G T A A T G C A T
NruI  (710)
1 site
T C G C G A A G C G C T
PshAI  (716)
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.
AarI  (854)
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.
SalI  (885)
1 site
G T C G A C C A G C T G
AccI  (886)
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.
SpeI  (893)
1 site
A C T A G T T G A T C A
TspMI  (901)
1 site
C C C G G G G G G C C C
XmaI  (901)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (903)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
PspOMI  (909)
1 site
G G G C C C C C C G G G
ApaI  (913)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BstBI  (918)
1 site
T T C G A A A A G C T T
BglII  (926)
2 sites
A G A T C T T C T A G A
EagI  (968)
1 site
C G G C C G G C C G G C
NotI  (968)
1 site
G C G G C C G C C G C C G G C G
HindIII  (979)
1 site
A A G C T T T T C G A A
Eco53kI  (989)
1 site
G A G C T C C T C G A G
SacI  (991)
1 site
G A G C T C C T C G A G
EcoRI  (995)
1 site
G A A T T C C T T A A G
Acc65I  (1007)
1 site
G G T A C C C C A T G G
KpnI  (1011)
1 site
G G T A C C C C A T G G
NcoI  (1015)
1 site
C C A T G G G G T A C C
BbsI  (1075)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
PvuII  (1091)
1 site
C A G C T G G T C G A C
BsrGI  (1507)
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  (1515)
1 site
C A C N N N N G T G G T G N N N N C A C
FspI  (1839)
1 site
T G C G C A A C G C G T
KasI  (1964)
1 site
G G C G C C C C G C G G
NarI  (1965)
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  (1966)
1 site
G G C G C C C C G C G G
PluTI  (1968)
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  (1971)
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  (2148)
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.
DraIII  (2163)
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  (2356)
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  (2397)
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.
XbaI  (2665)
1 site
T C T A G A A G A T C T
BseRI  (2668)
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  (2668)
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  (2705)
1 site
A T C G A T T A G C T A
ClaI  (2705)
1 site
A T C G A T T A G C T A
BamHI  (2714)
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  (2724)
1 site
A G G C C T T C C G G A
NheI  (2730)
1 site
G C T A G C C G A T C G
BmtI  (2734)
1 site
G C T A G C C G A T C G
AanI  (2880)
1 site
T T A T A A A A T A T T
PsiI  (2880)
1 site
T T A T A A A A T A T T
MfeI  (2909)
1 site
C A A T T G G T T A A C
luciferase
1017 .. 2669  =  1653 bp
550 amino acids  =  60.6 kDa
Product: firefly luciferase
synthetic luciferase gene
luciferase
1017 .. 2669  =  1653 bp
550 amino acids  =  60.6 kDa
Product: firefly luciferase
synthetic luciferase gene
NeoR/KanR
4903 .. 5697  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
4903 .. 5697  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
3569 .. 4157  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3569 .. 4157  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
298 .. 601  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
298 .. 601  =  304 bp
human cytomegalovirus immediate early enhancer
rrnG terminator
3091 .. 3227  =  137 bp
transcription terminator from the E. coli ribosomal
RNA rrnG operon (Albrechtsen et al., 1991)
rrnG terminator
3091 .. 3227  =  137 bp
transcription terminator from the E. coli ribosomal
RNA rrnG operon (Albrechtsen et al., 1991)
rrnG terminator
5710 .. 5846  =  137 bp
transcription terminator from the E. coli ribosomal
RNA rrnG operon (Albrechtsen et al., 1991)
rrnG terminator
5710 .. 5846  =  137 bp
transcription terminator from the E. coli ribosomal
RNA rrnG operon (Albrechtsen et al., 1991)
SV40 poly(A) signal
2779 .. 2900  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2779 .. 2900  =  122 bp
SV40 polyadenylation signal
pause site
793 .. 884  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
pause site
793 .. 884  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
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
3272 .. 3343  =  72 bp
insulator downstream of the human β-globin locus
(Farrell et al., 2002)
3' β-globin insulator
3272 .. 3343  =  72 bp
insulator downstream of the human β-globin locus
(Farrell et al., 2002)
poly(A) signal
731 .. 779  =  49 bp
synthetic polyadenylation signal
poly(A) signal
731 .. 779  =  49 bp
synthetic polyadenylation signal
T7 terminator
3015 .. 3061  =  47 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
3015 .. 3061  =  47 bp
transcription terminator for bacteriophage T7 RNA
polymerase
MCS
885 .. 922  =  38 bp
multiple cloning site
MCS
885 .. 922  =  38 bp
multiple cloning site
stop codons
2689 .. 2699  =  11 bp
stop codons in all three reading frames
stop codons
2689 .. 2699  =  11 bp
stop codons in all three reading frames
stop codons
2737 .. 2747  =  11 bp
stop codons in all three reading frames
stop codons
2737 .. 2747  =  11 bp
stop codons in all three reading frames
RBS
1003 .. 1008  =  6 bp
Shine-Dalgarno ribosome binding site
RBS
1003 .. 1008  =  6 bp
Shine-Dalgarno ribosome binding site
RBS
4890 .. 4895  =  6 bp
Shine-Dalgarno ribosome binding site
RBS
4890 .. 4895  =  6 bp
Shine-Dalgarno ribosome binding site
Kozak sequence
1013 .. 1019  =  7 bp
Kozak sequence
1013 .. 1019  =  7 bp
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