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Plasmid Files

pFC20A HaloTag® T7 SP6

Flexi® vector with an ampicillin resistance marker, for cell-free expression of a protein with a cleavable C-terminal HaloTag®.

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pFC20A HaloTag T7 SP6.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Promega
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AsiSI - PvuI - SgfI (75) BglII (3974) MluI (3855) BspEI (3642) PciI (2878) AgeI (2810) BstBI (2783) FspI (2485) BtgI - NcoI (78) Eco53kI - EcoICRI (459) SacI (461) PaeR7I - XhoI (462) BamHI (507) PflMI (548) AleI (606) BstEII (624) BseRI (626) PflFI - Tth111I (756) BmgBI (758) PasI (788) StuI * (957) BclI * (988) BspDI * - ClaI * (994) PshAI (1053) SgrAI (1140) BbsI (1339) BssHII (1365) EcoRI (1403) XbaI (1407) SalI (1413) PstI - SbfI (1423) BlpI (1488) EcoO109I (1556) SnaBI (1770) SspI (1903) XmnI (2108) pFC20A HaloTag® T7 SP6 4422 bp
AsiSI  (75)
1 site
G C G A T C G C C G C T A G C G
PvuI  (75)
1 site
C G A T C G G C T A G C
SgfI  (75)
1 site
G C G A T C G C C G C T A G C G
BglII  (3974)
1 site
A G A T C T T C T A G A
MluI  (3855)
1 site
A C G C G T T G C G C A
BspEI  (3642)
1 site
T C C G G A A G G C C T
PciI  (2878)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AgeI  (2810)
1 site
A C C G G T T G G C C A
BstBI  (2783)
1 site
T T C G A A A A G C T T
FspI  (2485)
1 site
T G C G C A A C G C G T
BtgI  (78)
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  (78)
1 site
C C A T G G G G T A C C
Eco53kI  (459)
1 site
G A G C T C C T C G A G
EcoICRI  (459)
1 site
G A G C T C C T C G A G
SacI  (461)
1 site
G A G C T C C T C G A G
PaeR7I  (462)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (462)
1 site
C T C G A G G A G C T C
BamHI  (507)
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  (548)
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  (606)
1 site
C A C N N N N G T G G T G N N N N C A C
BstEII  (624)
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  (626)
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  (756)
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  (756)
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  (758)
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  (788)
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  (957)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
BclI  (988)
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  (994)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (994)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
PshAI  (1053)
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  (1140)
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  (1339)
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  (1365)
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.
EcoRI  (1403)
1 site
G A A T T C C T T A A G
XbaI  (1407)
1 site
T C T A G A A G A T C T
SalI  (1413)
1 site
G T C G A C C A G C T G
PstI  (1423)
1 site
C T G C A G G A C G T C
SbfI  (1423)
1 site
C C T G C A G G G G A C G T C C
BlpI  (1488)
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  (1556)
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  (1770)
1 site
T A C G T A A T G C A T
SspI  (1903)
1 site
A A T A T T T T A T A A
XmnI  (2108)
1 site
G A A N N N N T T C C T T N N N N A A G
HaloTag®
513 .. 1394  =  882 bp
294 amino acids  =  33.4 kDa
Product: modified bacterial dehalogenase that forms covalent bonds with chloroalkane derivatives
HaloTag®
513 .. 1394  =  882 bp
294 amino acids  =  33.4 kDa
Product: modified bacterial dehalogenase that forms covalent bonds with chloroalkane derivatives
AmpR
1921 .. 2781  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1921 .. 1989  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1921 .. 2781  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1990 .. 2781  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1921 .. 2781  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2939 .. 3527  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2939 .. 3527  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
barnase
102 .. 437  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus amyloliquefaciens
The barnase gene is lethal in standard bacterial transformation strains.
barnase
102 .. 437  =  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
3643 .. 3926  =  284 bp
ColE1-derived recombination site that helps to maintain plasmids as monomers
cer region
3643 .. 3926  =  284 bp
ColE1-derived recombination site that helps to maintain plasmids as monomers
AmpR promoter
1816 .. 1920  =  105 bp
AmpR promoter
1816 .. 1920  =  105 bp
rrnB T1 terminator
4156 .. 4242  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
4156 .. 4242  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
T7 terminator
1540 .. 1587  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
1540 .. 1587  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
rrnB T2 terminator
4334 .. 4361  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
rrnB T2 terminator
4334 .. 4361  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
TEV site
477 .. 497  =  21 bp
7 amino acids  =  901.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV protease recognition site
TEV site
477 .. 497  =  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
SP6 promoter
45 .. 63  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
45 .. 63  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
ORF:  1921 .. 2781  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  102 .. 437  =  336 bp
ORF:  111 amino acids  =  12.5 kDa
ORF:  444 .. 1400  =  957 bp
ORF:  318 amino acids  =  36.0 kDa
ORF:  3888 .. 4235  =  348 bp
ORF:  115 amino acids  =  12.4 kDa
ORF:  3997 .. 4323  =  327 bp
ORF:  108 amino acids  =  12.1 kDa
ORF:  441 .. 1070  =  630 bp
ORF:  209 amino acids  =  23.0 kDa
ORF:  2385 .. 2651  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1070 .. 1306  =  237 bp
ORF:  78 amino acids  =  8.0 kDa
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