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pFC14K HaloTag® CMV

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

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pFC14K HaloTag CMV Sequence and MappFC14K HaloTag CMV.dna
Map and Sequence File   
Sequence Author:  Promega
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 ScaI (5011) MluI (4927) BspEI (4714) BspHI (4670) ApaLI (4264) PciI (3950) AgeI (3882) BstBI (3855) RsrII (3689) NaeI (3675) NgoMIV (3673) FspI (3275) XcmI (2989) MfeI (2655) HpaI (2646) AanI - AanI - PsiI (2626) BsrGI (96) SpeI (152) NdeI (387) SnaBI (493) HindIII (748) T7 promoter NheI (1052) BmtI (1056) AsiSI - PvuI - SgfI (1060) AhdI (1436) Eco53kI - EcoICRI (1444) SacI (1446) PaeR7I - XhoI (1447) PflMI (1533) AleI (1591) BstEII (1609) BseRI (1611) BmgBI (1743) PasI (1773) StuI * (1942) BspDI * - ClaI * (1979) AclI (1988) PshAI (2038) SgrAI (2125) BssHII (2350) XbaI (2392) SalI (2398) SbfI (2408) BlpI (2473) NotI (2503) pFC14K HaloTag® CMV 5051 bp
ScaI  (5011)
1 site
A G T A C T T C A T G A
MluI  (4927)
1 site
A C G C G T T G C G C A
BspEI  (4714)
1 site
T C C G G A A G G C C T
BspHI  (4670)
1 site
T C A T G A A G T A C T
ApaLI  (4264)
1 site
G T G C A C C A C G T G
PciI  (3950)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AgeI  (3882)
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  (3855)
1 site
T T C G A A A A G C T T
RsrII  (3689)
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.
NaeI  (3675)
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.
NgoMIV  (3673)
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.
FspI  (3275)
1 site
T G C G C A A C G C G T
XcmI  (2989)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
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
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
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.
SnaBI  (493)
1 site
T A C G T A A T G C A T
HindIII  (748)
1 site
A A G C T T T T C G A A
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
AhdI  (1436)
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.
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.
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.
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.
AclI  (1988)
1 site
A A C G T T T T G C A A
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
NeoR/KanR
3045 .. 3839  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
3045 .. 3839  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
4011 .. 4599  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4011 .. 4599  =  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
4715 .. 4998  =  284 bp
ColE1-derived recombination site that helps to
maintain plasmids as monomers
cer region
4715 .. 4998  =  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
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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