pFC27K HaloTag CMV-neo

Flexi® vector with a kanamycin/G418 resistance marker, for mammalian expression of a protein with a cleavable C-terminal HaloTag®.

Sequence Author: Promega

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ScaI (5487) MluI (5403) BspEI (5190) ApaLI (4740) PciI (4426) BstXI (4191) BstBI (4152) RsrII (3986) NaeI (3972) NgoMIV (3970) PluTI (3473) SfoI (3471) NarI (3470) KasI (3469) PacI (3257) AvrII (3233) SfiI (3186) SexAI * (3000) KpnI (2887) Acc65I (2883) BglII (5522) BsrGI (96) SpeI (152) CMV enhancer NdeI (387) SnaBI (493) 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) BmgBI (1743) PasI (1773) BclI * (1973) AclI (1988) PshAI (2038) SgrAI (2125) EcoRI (2388) XbaI (2392) SalI (2398) SbfI (2408) BlpI (2473) NotI (2503) AanI - AanI - PsiI (2626) HpaI (2646) MfeI (2655) pFC27K HaloTag® CMV-neo 5527 bp
ScaI  (5487)
1 site
A G T A C T T C A T G A
MluI  (5403)
1 site
A C G C G T T G C G C A
BspEI  (5190)
1 site
T C C G G A A G G C C T
ApaLI  (4740)
1 site
G T G C A C C A C G T G
PciI  (4426)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstXI  (4191)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
BstBI  (4152)
1 site
T T C G A A A A G C T T
RsrII  (3986)
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  (3972)
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  (3970)
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.
PluTI  (3473)
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.
SfoI  (3471)
1 site
G G C G C C C C G C G G
NarI  (3470)
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.
KasI  (3469)
1 site
G G C G C C C C G C G G
PacI  (3257)
1 site
T T A A T T A A A A T T A A T T
AvrII  (3233)
1 site
C C T A G G G G A T C C
SfiI  (3186)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
SexAI  (3000)
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.
KpnI  (2887)
1 site
G G T A C C C C A T G G
Acc65I  (2883)
1 site
G G T A C C C C A T G G
BglII  (5522)
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.
SnaBI  (493)
1 site
T A C G T A A T G C A T
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.
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.
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.
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.
EcoRI  (2388)
1 site
G A A T T C C T T A A G
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
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
HpaI  (2646)
1 site
G T T A A C C A A T T G
MfeI  (2655)
1 site
C A A T T G G T T A A C
TEV site
1462 .. 1482  =  21 bp
7 amino acids  =  900.9 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV protease recognition site
TEV site
1462 .. 1482  =  21 bp
7 amino acids  =  900.9 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV protease recognition site
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
3342 .. 4136  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
3342 .. 4136  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
ori
4487 .. 5075  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4487 .. 5075  =  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
SV40 promoter
2891 .. 3248  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
2891 .. 3248  =  358 bp
SV40 enhancer and early promoter
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
5191 .. 5474  =  284 bp
ColE1-derived recombination site that helps to maintain plasmids as monomers
cer region
5191 .. 5474  =  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
poly(A) signal
4200 .. 4248  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4200 .. 4248  =  49 bp
synthetic polyadenylation signal
EM7 promoter
3263 .. 3310  =  48 bp
synthetic bacterial promoter
EM7 promoter
3263 .. 3310  =  48 bp
synthetic bacterial promoter
T7 promoter
1033 .. 1051  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1033 .. 1051  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SV40 ori
3099 .. 3234  =  136 bp
SV40 origin of replication
SV40 ori
3099 .. 3234  =  136 bp
SV40 origin of replication
ORF:  1087 .. 1422  =  336 bp
ORF:  111 amino acids  =  12.5 kDa
ORF:  1429 .. 2385  =  957 bp
ORF:  318 amino acids  =  36.0 kDa
ORF:  3514 .. 3900  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  3342 .. 4136  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  821 .. 1066  =  246 bp
ORF:  81 amino acids  =  8.6 kDa
ORF:  1426 .. 2055  =  630 bp
ORF:  209 amino acids  =  23.0 kDa
ORF:  4012 .. 4317  =  306 bp
ORF:  101 amino acids  =  11.5 kDa
ORF:  2055 .. 2291  =  237 bp
ORF:  78 amino acids  =  8.0 kDa
ORF:  3651 .. 4187  =  537 bp
ORF:  178 amino acids  =  19.8 kDa
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