pFN29A His6HaloTag T7

Flexi® vector with an ampicillin resistance marker, for bacterial or cell-free expression of a protein with a cleavable N-terminal 6xHis-HaloTag® dual tag.

Sequence Author: Promega

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T7 promoter BglII (3950) MluI (3831) BspEI (3618) PciI (2854) AgeI (2786) BstBI (2759) AhdI (2684) FspI (2461) ATG 6xHis PflMI (132) AleI (190) BstEII (208) BseRI (210) PflFI - Tth111I (340) BmgBI (342) PasI (372) StuI * (541) BclI * (572) BspDI * - ClaI * (578) PshAI (637) SgrAI (724) BbsI (923) BssHII (949) PaeR7I - XhoI (967) AsiSI - PvuI - SgfI (1025) BtgI - NcoI (1028) DraI - PmeI (1392) Acc65I (1409) KpnI (1413) BamHI (1418) XbaI (1424) PstI - SbfI (1440) BlpI (1505) EcoO109I (1532) SnaBI (1746) SspI (1879) XmnI (2084) pFN29A His6HaloTag® T7 4398 bp
BglII  (3950)
1 site
A G A T C T T C T A G A
MluI  (3831)
1 site
A C G C G T T G C G C A
BspEI  (3618)
1 site
T C C G G A A G G C C T
PciI  (2854)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AgeI  (2786)
1 site
A C C G G T T G G C C A
BstBI  (2759)
1 site
T T C G A A A A G C T T
AhdI  (2684)
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  (2461)
1 site
T G C G C A A C G C G T
PflMI  (132)
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  (190)
1 site
C A C N N N N G T G G T G N N N N C A C
BstEII  (208)
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  (210)
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  (340)
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  (340)
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  (342)
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  (372)
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  (541)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
BclI  (572)
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  (578)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (578)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
PshAI  (637)
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  (724)
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.
BbsI  (923)
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.
BssHII  (949)
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.
PaeR7I  (967)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (967)
1 site
C T C G A G G A G C T C
AsiSI  (1025)
1 site
G C G A T C G C C G C T A G C G
PvuI  (1025)
1 site
C G A T C G G C T A G C
SgfI  (1025)
1 site
G C G A T C G C C G C T A G C G
BtgI  (1028)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (1028)
1 site
C C A T G G G G T A C C
DraI  (1392)
1 site
T T T A A A A A A T T T
PmeI  (1392)
1 site
G T T T A A A C C A A A T T T G
Acc65I  (1409)
1 site
G G T A C C C C A T G G
KpnI  (1413)
1 site
G G T A C C C C A T G G
BamHI  (1418)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
XbaI  (1424)
1 site
T C T A G A A G A T C T
PstI  (1440)
1 site
C T G C A G G A C G T C
SbfI  (1440)
1 site
C C T G C A G G G G A C G T C C
BlpI  (1505)
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.
EcoO109I  (1532)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
SnaBI  (1746)
1 site
T A C G T A A T G C A T
SspI  (1879)
1 site
A A T A T T T T A T A A
XmnI  (2084)
1 site
G A A N N N N T T C C T T N N N N A A G
ATG
70 .. 72  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
70 .. 72  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
76 .. 93  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
76 .. 93  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
HaloTag®
94 .. 981  =  888 bp
296 amino acids  =  33.5 kDa
Product: modified bacterial dehalogenase that forms covalent bonds with chloroalkane derivatives
HaloTag®
94 .. 981  =  888 bp
296 amino acids  =  33.5 kDa
Product: modified bacterial dehalogenase that forms covalent bonds with chloroalkane derivatives
TEV site
994 .. 1014  =  21 bp
7 amino acids  =  900.9 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV protease recognition site
TEV site
994 .. 1014  =  21 bp
7 amino acids  =  900.9 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV protease recognition site
AmpR
1897 .. 2757  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   1897 .. 1965  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1897 .. 2757  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   1966 .. 2757  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1897 .. 2757  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2915 .. 3503  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2915 .. 3503  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
barnase
1052 .. 1387  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus amyloliquefaciens
The barnase gene is lethal in standard bacterial transformation strains.
barnase
1052 .. 1387  =  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
3619 .. 3902  =  284 bp
ColE1-derived recombination site that helps to maintain plasmids as monomers
cer region
3619 .. 3902  =  284 bp
ColE1-derived recombination site that helps to maintain plasmids as monomers
AmpR promoter
1792 .. 1896  =  105 bp
AmpR promoter
1792 .. 1896  =  105 bp
rrnB T1 terminator
4132 .. 4218  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
4132 .. 4218  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
T7 terminator
1516 .. 1563  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
1516 .. 1563  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
rrnB T2 terminator
4310 .. 4337  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
rrnB T2 terminator
4310 .. 4337  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
T7 promoter
21 .. 39  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
21 .. 39  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ORF:  70 .. 1038  =  969 bp
ORF:  322 amino acids  =  36.6 kDa
ORF:  1897 .. 2757  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  1052 .. 1387  =  336 bp
ORF:  111 amino acids  =  12.5 kDa
ORF:  3864 .. 4211  =  348 bp
ORF:  115 amino acids  =  12.4 kDa
ORF:  4369 .. 1053  =  1083 bp
ORF:  360 amino acids  =  40.8 kDa
ORF:  3973 .. 4299  =  327 bp
ORF:  108 amino acids  =  12.1 kDa
ORF:  654 .. 890  =  237 bp
ORF:  78 amino acids  =  8.0 kDa
ORF:  2361 .. 2627  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  869 .. 1108  =  240 bp
ORF:  79 amino acids  =  8.9 kDa
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Download pFN29A His6HaloTag T7.dna file

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