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pF4A CMV

Flexi® vector with an ampicillin resistance marker, for protein expression in mammalian cells.

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pF4A CMV Sequence and MappF4A CMV.dna
Map and Sequence File   
Sequence Author:  Promega
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 BsaBI * (4117) MluI (3999) BspEI (3786) HaeII (3270) PciI (3022) AgeI (2954) BstBI (2927) AhdI (2852) FspI (2629) XmnI (2252) EarI (2053) BglII (4118) BsrGI (96) SpeI (152) CMV enhancer NdeI (387) BtgZI (487) HindIII (748) BstAPI (794) BbsI (928) T7 promoter NheI (1052) BmtI (1056) AsiSI - PvuI - SgfI (1060) barnase DraI - PmeI (1427) Eco53kI (1440) BanII - SacI (1442) Acc65I (1444) AvaI - BsoBI - KpnI - TspMI - XmaI (1448) BmeT110I (1449) SmaI (1450) XbaI (1459) SalI (1465) AccI (1466) SbfI (1475) SphI (1481) BlpI (1540) EagI - NotI (1570) PsiI (1693) HpaI (1713) MfeI (1722) BsrBI (2010) AmpR promoter pF4A CMV 4123 bp
BsaBI  (4117)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
MluI  (3999)
1 site
A C G C G T T G C G C A
BspEI  (3786)
1 site
T C C G G A A G G C C T
HaeII  (3270)
1 site
R G C G C Y Y C G C G R
PciI  (3022)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AgeI  (2954)
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.
BstBI  (2927)
1 site
T T C G A A A A G C T T
AhdI  (2852)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
FspI  (2629)
1 site
T G C G C A A C G C G T
XmnI  (2252)
1 site
G A A N N N N T T C C T T N N N N A A G
EarI  (2053)
1 site
C T C T T C N G A G A A G N N N N

Efficient cleavage requires at least two copies of the EarI
recognition sequence.
Sticky ends from different EarI sites may not be compatible.
BglII  (4118)
1 site
A G A T C T T C T A G A
BsrGI  (96)
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.
SpeI  (152)
1 site
A C T A G T T G A T C A
NdeI  (387)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
BtgZI  (487)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its
electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
HindIII  (748)
1 site
A A G C T T T T C G A A
BstAPI  (794)
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.
BbsI  (928)
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.
NheI  (1052)
1 site
G C T A G C C G A T C G
BmtI  (1056)
1 site
G C T A G C C G A T C G
AsiSI  (1060)
1 site
G C G A T C G C C G C T A G C G
PvuI  (1060)
1 site
C G A T C G G C T A G C
SgfI  (1060)
1 site
G C G A T C G C C G C T A G C G
DraI  (1427)
1 site
T T T A A A A A A T T T
PmeI  (1427)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (1440)
1 site
G A G C T C C T C G A G
BanII  (1442)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
SacI  (1442)
1 site
G A G C T C C T C G A G
Acc65I  (1444)
1 site
G G T A C C C C A T G G
AvaI  (1448)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (1448)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
KpnI  (1448)
1 site
G G T A C C C C A T G G
TspMI  (1448)
1 site
C C C G G G G G G C C C
XmaI  (1448)
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.
BmeT110I  (1449)
1 site
C Y C G R G G R G C Y C
SmaI  (1450)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
XbaI  (1459)
1 site
T C T A G A A G A T C T
SalI  (1465)
1 site
G T C G A C C A G C T G
AccI  (1466)
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.
SbfI  (1475)
1 site
C C T G C A G G G G A C G T C C
SphI  (1481)
1 site
G C A T G C C G T A C G
BlpI  (1540)
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.
EagI  (1570)
1 site
C G G C C G G C C G G C
NotI  (1570)
1 site
G C G G C C G C C G C C G G C G
PsiI  (1693)
1 site
T T A T A A A A T A T T
HpaI  (1713)
1 site
G T T A A C C A A T T G
MfeI  (1722)
1 site
C A A T T G G T T A A C
BsrBI  (2010)
1 site
C C G C T C G G C G A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BsrBI will not always regenerate a BsrBI site.
BsrBI is typically used at 37°C, but can be used at temperatures
up to 50°C.
AmpR
2065 .. 2925  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2065 .. 2133  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2065 .. 2925  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2134 .. 2925  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2065 .. 2925  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3083 .. 3671  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3083 .. 3671  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
138 .. 517  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
138 .. 517  =  380 bp
human cytomegalovirus immediate early enhancer
barnase
1087 .. 1422  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
barnase
1087 .. 1422  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
cer region
3787 .. 4070  =  284 bp
ColE1-derived recombination site that helps to
maintain plasmids as monomers
cer region
3787 .. 4070  =  284 bp
ColE1-derived recombination site that helps to
maintain plasmids as monomers
CMV promoter
518 .. 721  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
518 .. 721  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
chimeric intron
857 .. 989  =  133 bp
chimera between introns from human β-globin and
immunoglobulin heavy chain genes
chimeric intron
857 .. 989  =  133 bp
chimera between introns from human β-globin and
immunoglobulin heavy chain genes
SV40 poly(A) signal
1592 .. 1713  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1592 .. 1713  =  122 bp
SV40 polyadenylation signal
AmpR promoter
1960 .. 2064  =  105 bp
AmpR promoter
1960 .. 2064  =  105 bp
T7 promoter
1033 .. 1051  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1033 .. 1051  =  19 bp
promoter for bacteriophage T7 RNA polymerase
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