pFN28A HaloTag CMV-neo

Flexi® vector with ampicillin and G418 resistance markers, for mammalian expression of a protein with a cleavable N-terminal HaloTag®.

Sequence Author: Promega

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BglII (6527) MluI (6408) BspEI (6195) PciI (5431) AgeI (5363) AhdI (5261) XmnI (4661) poly(A) signal BstXI (4156) RsrII (3951) NaeI (3937) NgoMIV (3935) PluTI (3438) SfoI (3436) NarI (3435) KasI (3434) BsrGI (96) SpeI (152) CMV enhancer NdeI (387) T7 promoter NheI (1052) BmtI (1056) PflMI (1108) AleI (1166) BstEII (1184) BmgBI (1318) PasI (1348) BclI * (1548) PshAI (1613) SgrAI (1700) PaeR7I - XhoI (1943) AsiSI - PvuI - SgfI (2001) DraI - PmeI (2368) EcoRI (2373) XbaI (2400) SbfI (2416) BlpI (2481) NotI (2511) AanI - PsiI (2634) HpaI (2654) MfeI (2663) SexAI * (3008) SfiI (3194) AvrII (3241) pFN28A HaloTag® CMV-neo 6532 bp
BglII  (6527)
1 site
A G A T C T T C T A G A
MluI  (6408)
1 site
A C G C G T T G C G C A
BspEI  (6195)
1 site
T C C G G A A G G C C T
PciI  (5431)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AgeI  (5363)
1 site
A C C G G T T G G C C A
AhdI  (5261)
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.
XmnI  (4661)
1 site
G A A N N N N T T C C T T N N N N A A G
BstXI  (4156)
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.
RsrII  (3951)
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  (3937)
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  (3935)
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  (3438)
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  (3436)
1 site
G G C G C C C C G C G G
NarI  (3435)
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  (3434)
1 site
G G C G C C C C G C G G
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.
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
PflMI  (1108)
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  (1166)
1 site
C A C N N N N G T G G T G N N N N C A C
BstEII  (1184)
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  (1318)
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  (1348)
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  (1548)
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.
PshAI  (1613)
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  (1700)
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.
PaeR7I  (1943)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1943)
1 site
C T C G A G G A G C T C
AsiSI  (2001)
1 site
G C G A T C G C C G C T A G C G
PvuI  (2001)
1 site
C G A T C G G C T A G C
SgfI  (2001)
1 site
G C G A T C G C C G C T A G C G
DraI  (2368)
1 site
T T T A A A A A A T T T
PmeI  (2368)
1 site
G T T T A A A C C A A A T T T G
EcoRI  (2373)
1 site
G A A T T C C T T A A G
XbaI  (2400)
1 site
T C T A G A A G A T C T
SbfI  (2416)
1 site
C C T G C A G G G G A C G T C C
BlpI  (2481)
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  (2511)
1 site
G C G G C C G C C G C C G G C G
AanI  (2634)
1 site
T T A T A A A A T A T T
PsiI  (2634)
1 site
T T A T A A A A T A T T
HpaI  (2654)
1 site
G T T A A C C A A T T G
MfeI  (2663)
1 site
C A A T T G G T T A A C
SexAI  (3008)
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.
SfiI  (3194)
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.
AvrII  (3241)
1 site
C C T A G G G G A T C C
HaloTag®
1067 .. 1957  =  891 bp
297 amino acids  =  33.6 kDa
Product: modified bacterial dehalogenase that forms covalent bonds with chloroalkane derivatives
HaloTag®
1067 .. 1957  =  891 bp
297 amino acids  =  33.6 kDa
Product: modified bacterial dehalogenase that forms covalent bonds with chloroalkane derivatives
TEV site
1970 .. 1990  =  21 bp
7 amino acids  =  900.9 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV protease recognition site
TEV site
1970 .. 1990  =  21 bp
7 amino acids  =  900.9 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV protease recognition site
AmpR
4474 .. 5334  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   4474 .. 4542  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4474 .. 5334  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   4543 .. 5334  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4474 .. 5334  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
3307 .. 4101  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
3307 .. 4101  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
ori
5492 .. 6080  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5492 .. 6080  =  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
2899 .. 3256  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
2899 .. 3256  =  358 bp
SV40 enhancer and early promoter
barnase
2028 .. 2363  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus amyloliquefaciens
The barnase gene is lethal in standard bacterial transformation strains.
barnase
2028 .. 2363  =  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
6196 .. 6479  =  284 bp
ColE1-derived recombination site that helps to maintain plasmids as monomers
cer region
6196 .. 6479  =  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
2533 .. 2654  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2533 .. 2654  =  122 bp
SV40 polyadenylation signal
AmpR promoter
4369 .. 4473  =  105 bp
AmpR promoter
4369 .. 4473  =  105 bp
poly(A) signal
4165 .. 4213  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4165 .. 4213  =  49 bp
synthetic polyadenylation signal
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
3107 .. 3242  =  136 bp
SV40 origin of replication
SV40 ori
3107 .. 3242  =  136 bp
SV40 origin of replication
ORF:  3307 .. 4101  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  4474 .. 5334  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  1067 .. 2014  =  948 bp
ORF:  315 amino acids  =  35.7 kDa
ORF:  3479 .. 3865  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  2028 .. 2363  =  336 bp
ORF:  111 amino acids  =  12.5 kDa
ORF:  821 .. 2029  =  1209 bp
ORF:  402 amino acids  =  44.8 kDa
ORF:  3977 .. 4282  =  306 bp
ORF:  101 amino acids  =  11.5 kDa
ORF:  1630 .. 1866  =  237 bp
ORF:  78 amino acids  =  8.0 kDa
ORF:  3616 .. 4152  =  537 bp
ORF:  178 amino acids  =  19.8 kDa
ORF:  1845 .. 2084  =  240 bp
ORF:  79 amino acids  =  8.9 kDa
ORF:  4938 .. 5204  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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