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pFC30A His6HaloTag® T7

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

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pFC30A His6HaloTag T7.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Promega
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AsiSI - PvuI - SgfI (65) BglII (3864) MluI (3745) BspEI (3532) PciI (2768) AgeI (2700) BstBI (2673) FspI (2375) BtgI - NcoI (68) Eco53kI - EcoICRI (449) SacI (451) PaeR7I - XhoI (452) BamHI (497) PflMI (538) SphI (562) AleI (596) BstEII (614) BseRI (616) PflFI - Tth111I (746) BmgBI (748) PasI (778) StuI * (947) BclI * (978) BspDI * - ClaI * (984) PshAI (1043) SgrAI (1130) BbsI (1329) BssHII (1355) AccI (1367) BlpI (1419) EcoO109I (1446) BfuAI - BspMI (1571) SnaBI (1660) SspI (1793) XmnI (1998) pFC30A His6HaloTag® T7 4312 bp
AsiSI  (65)
1 site
G C G A T C G C C G C T A G C G
PvuI  (65)
1 site
C G A T C G G C T A G C
SgfI  (65)
1 site
G C G A T C G C C G C T A G C G
BglII  (3864)
1 site
A G A T C T T C T A G A
MluI  (3745)
1 site
A C G C G T T G C G C A
BspEI  (3532)
1 site
T C C G G A A G G C C T
PciI  (2768)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AgeI  (2700)
1 site
A C C G G T T G G C C A
BstBI  (2673)
1 site
T T C G A A A A G C T T
FspI  (2375)
1 site
T G C G C A A C G C G T
BtgI  (68)
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  (68)
1 site
C C A T G G G G T A C C
Eco53kI  (449)
1 site
G A G C T C C T C G A G
EcoICRI  (449)
1 site
G A G C T C C T C G A G
SacI  (451)
1 site
G A G C T C C T C G A G
PaeR7I  (452)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (452)
1 site
C T C G A G G A G C T C
BamHI  (497)
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.
PflMI  (538)
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.
SphI  (562)
1 site
G C A T G C C G T A C G
AleI  (596)
1 site
C A C N N N N G T G G T G N N N N C A C
BstEII  (614)
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  (616)
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  (746)
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  (746)
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  (748)
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  (778)
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  (947)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
BclI  (978)
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  (984)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (984)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
PshAI  (1043)
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  (1130)
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  (1329)
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  (1355)
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.
AccI  (1367)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
BlpI  (1419)
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  (1446)
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.
BfuAI  (1571)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (1571)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
SnaBI  (1660)
1 site
T A C G T A A T G C A T
SspI  (1793)
1 site
A A T A T T T T A T A A
XmnI  (1998)
1 site
G A A N N N N T T C C T T N N N N A A G
HaloTag®
503 .. 1384  =  882 bp
294 amino acids  =  33.4 kDa
Product: modified bacterial dehalogenase that forms covalent bonds with chloroalkane derivatives
HaloTag®
503 .. 1384  =  882 bp
294 amino acids  =  33.4 kDa
Product: modified bacterial dehalogenase that forms covalent bonds with chloroalkane derivatives
AmpR
1811 .. 2671  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1811 .. 1879  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1811 .. 2671  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1880 .. 2671  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1811 .. 2671  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2829 .. 3417  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2829 .. 3417  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
barnase
92 .. 427  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus amyloliquefaciens
The barnase gene is lethal in standard bacterial transformation strains.
barnase
92 .. 427  =  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
3533 .. 3816  =  284 bp
ColE1-derived recombination site that helps to maintain plasmids as monomers
cer region
3533 .. 3816  =  284 bp
ColE1-derived recombination site that helps to maintain plasmids as monomers
AmpR promoter
1706 .. 1810  =  105 bp
AmpR promoter
1706 .. 1810  =  105 bp
rrnB T1 terminator
4046 .. 4132  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
4046 .. 4132  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
T7 terminator
1430 .. 1477  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
1430 .. 1477  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
rrnB T2 terminator
4224 .. 4251  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
rrnB T2 terminator
4224 .. 4251  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
TEV site
467 .. 487  =  21 bp
7 amino acids  =  901.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV protease recognition site
TEV site
467 .. 487  =  21 bp
7 amino acids  =  901.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV protease recognition site
T7 promoter
21 .. 39  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
21 .. 39  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
1388 .. 1405  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1388 .. 1405  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ORF:  3778 .. 4125  =  348 bp
ORF:  115 amino acids  =  12.4 kDa
ORF:  92 .. 427  =  336 bp
ORF:  111 amino acids  =  12.5 kDa
ORF:  434 .. 1408  =  975 bp
ORF:  324 amino acids  =  36.8 kDa
ORF:  1811 .. 2671  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  431 .. 1399  =  969 bp
ORF:  322 amino acids  =  36.1 kDa
ORF:  3887 .. 4213  =  327 bp
ORF:  108 amino acids  =  12.1 kDa
ORF:  1060 .. 1296  =  237 bp
ORF:  78 amino acids  =  8.0 kDa
ORF:  2275 .. 2541  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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