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Plasmid Files

pFC20K HaloTag® T7 SP6

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

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pFC20K HaloTag T7 SP6 Sequence and MappFC20K HaloTag T7 SP6.dna
Map and Sequence File   
Sequence Author:  Promega
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 AseI (20) ScaI (3934) MluI (3850) BspEI (3637) BspHI (3593) ApaLI (3187) PciI (2873) AgeI (2805) BstBI (2778) RsrII (2612) NaeI (2598) NgoMIV (2596) T7 promoter SP6 promoter AsiSI - PvuI - SgfI (75) BtgI - NcoI (78) AhdI (451) Eco53kI - EcoICRI (459) SacI (461) PaeR7I - XhoI (462) BamHI (507) PflMI (548) AleI (606) BstEII (624) BseRI (626) BmgBI (758) PasI (788) StuI * (957) BspDI * - ClaI * (994) PshAI (1053) BglI (1112) SgrAI (1140) BbsI (1339) BssHII (1365) EcoRI (1403) XbaI (1407) SalI (1413) SbfI (1423) BlpI (1488) EcoO109I (1556) Bts α I (1660) BstAPI (1794) XcmI (1912) FspI (2198) pFC20K HaloTag® T7 SP6 4417 bp
AseI  (20)
1 site
A T T A A T T A A T T A
ScaI  (3934)
1 site
A G T A C T T C A T G A
MluI  (3850)
1 site
A C G C G T T G C G C A
BspEI  (3637)
1 site
T C C G G A A G G C C T
BspHI  (3593)
1 site
T C A T G A A G T A C T
ApaLI  (3187)
1 site
G T G C A C C A C G T G
PciI  (2873)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AgeI  (2805)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BstBI  (2778)
1 site
T T C G A A A A G C T T
RsrII  (2612)
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  (2598)
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  (2596)
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.
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
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
AhdI  (451)
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  (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.
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.
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.
BglI  (1112)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
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
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.
BtsαI  (1660)
1 site
G C A G T G N N C G T C A C

Sticky ends from different BtsI sites may not be compatible.
BstAPI  (1794)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
XcmI  (1912)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
FspI  (2198)
1 site
T G C G C A A C G C G T
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
NeoR/KanR
1968 .. 2762  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
1968 .. 2762  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
2934 .. 3522  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2934 .. 3522  =  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
3638 .. 3921  =  284 bp
ColE1-derived recombination site that helps to
maintain plasmids as monomers
cer region
3638 .. 3921  =  284 bp
ColE1-derived recombination site that helps to
maintain plasmids as monomers
rrnB T1 terminator
4151 .. 4237  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
4151 .. 4237  =  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
4329 .. 4356  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
rrnB T2 terminator
4329 .. 4356  =  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
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