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pFC14A HaloTag® CMV

Flexi® vector with an ampicillin resistance marker, for mammalian expression of a protein with a cleavable C-terminal HaloTag®.

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pFC14A HaloTag CMV Sequence and MappFC14A HaloTag CMV.dna
Map and Sequence File   
Sequence Author:  Promega
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 BsaBI * (5050) MluI (4932) BspEI (4719) PciI (3955) AgeI (3887) BstBI (3860) FspI (3562) XmnI (3185) MfeI (2655) HpaI (2646) AanI - PsiI (2626) BglII (5051) BsrGI (96) SpeI (152) NdeI (387) HindIII (748) BstAPI (794) T7 promoter NheI (1052) BmtI (1056) AsiSI - PvuI - SgfI (1060) Eco53kI - EcoICRI (1444) SacI (1446) PaeR7I - XhoI (1447) PflMI (1533) AleI (1591) BstEII (1609) BseRI (1611) PflFI - Tth111I (1741) BmgBI (1743) PasI (1773) StuI * (1942) BclI * (1973) BspDI * - ClaI * (1979) PshAI (2038) SgrAI (2125) BssHII (2350) XbaI (2392) SalI (2398) SbfI (2408) BlpI (2473) NotI (2503) pFC14A HaloTag® CMV 5056 bp
BsaBI  (5050)
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  (4932)
1 site
A C G C G T T G C G C A
BspEI  (4719)
1 site
T C C G G A A G G C C T
PciI  (3955)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AgeI  (3887)
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  (3860)
1 site
T T C G A A A A G C T T
FspI  (3562)
1 site
T G C G C A A C G C G T
XmnI  (3185)
1 site
G A A N N N N T T C C T T N N N N A A G
MfeI  (2655)
1 site
C A A T T G G T T A A C
HpaI  (2646)
1 site
G T T A A C C A A T T G
AanI  (2626)
1 site
T T A T A A A A T A T T
PsiI  (2626)
1 site
T T A T A A A A T A T T
BglII  (5051)
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.
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.
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
Eco53kI  (1444)
1 site
G A G C T C C T C G A G
EcoICRI  (1444)
1 site
G A G C T C C T C G A G
SacI  (1446)
1 site
G A G C T C C T C G A G
PaeR7I  (1447)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1447)
1 site
C T C G A G G A G C T C
PflMI  (1533)
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.
AleI  (1591)
1 site
C A C N N N N G T G G T G N N N N C A C
BstEII  (1609)
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.
BseRI  (1611)
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.
PflFI  (1741)
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  (1741)
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.
BmgBI  (1743)
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.
PasI  (1773)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
StuI  (1942)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
BclI  (1973)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
BspDI  (1979)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1979)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
PshAI  (2038)
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.
SgrAI  (2125)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
BssHII  (2350)
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.
XbaI  (2392)
1 site
T C T A G A A G A T C T
SalI  (2398)
1 site
G T C G A C C A G C T G
SbfI  (2408)
1 site
C C T G C A G G G G A C G T C C
BlpI  (2473)
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.
NotI  (2503)
1 site
G C G G C C G C C G C C G G C G
HaloTag®
1498 .. 2379  =  882 bp
294 amino acids  =  33.4 kDa
Product: modified bacterial dehalogenase that forms
covalent bonds with chloroalkane derivatives
HaloTag®
1498 .. 2379  =  882 bp
294 amino acids  =  33.4 kDa
Product: modified bacterial dehalogenase that forms
covalent bonds with chloroalkane derivatives
AmpR
2998 .. 3858  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2998 .. 3066  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2998 .. 3858  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3067 .. 3858  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2998 .. 3858  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
4016 .. 4604  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4016 .. 4604  =  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
4720 .. 5003  =  284 bp
ColE1-derived recombination site that helps to
maintain plasmids as monomers
cer region
4720 .. 5003  =  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
2525 .. 2646  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2525 .. 2646  =  122 bp
SV40 polyadenylation signal
AmpR promoter
2893 .. 2997  =  105 bp
AmpR promoter
2893 .. 2997  =  105 bp
TEV site
1462 .. 1482  =  21 bp
7 amino acids  =  901.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV protease recognition site
TEV site
1462 .. 1482  =  21 bp
7 amino acids  =  901.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV protease recognition site
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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