pQE-100 DoubleTag

Bacterial vector for expressing proteins with an N-terminal 6xHis tag and a C-terminal Tag-100 tag.

Sequence Author: Qiagen

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MfeI (59) PsiI (49) T5 promoter PaeR7I - XhoI (1) BbsI (3498) EcoO109I (3496) AatII (3442) ZraI (3440) XmnI (3119) PvuI (2890) FspI (2742) AseI (2692) NmeAIII (2668) BglI (2640) BsrFI (2600) BsaI (2581) AhdI (2520) AlwNI (2043) PspFI (1935) BseYI (1931) lac operator EcoRI (88) RBS BseRI (115) ATG 6xHis BamHI (145) SphI (155) Eco53kI (159) BanII - SacI (161) Acc65I (163) KpnI (167) TspMI - XmaI (168) SmaI (170) SalI (173) HincII (175) PstI (183) BsgI (185) BfuAI - BspMI (186) Tag-100 BglII (232) stop codons BlpI (250) NheI (358) BmtI (362) Bpu10I (383) PvuII (510) BspEI (606) PasI - PflMI * (842) MscI (877) BtgI - NcoI - StyI (911) XbaI (1214) PfoI (1271) PflFI - Tth111I (1374) BsaAI (1381) BstZ17I (1400) NdeI (1450) BstAPI (1451) BspQI - SapI (1511) AflIII - PciI (1627) pQE-100 DoubleTag™ 3512 bp
MfeI  (59)
1 site
C A A T T G G T T A A C
PsiI  (49)
1 site
T T A T A A A A T A T T
PaeR7I  (1)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1)
1 site
C T C G A G G A G C T C
BbsI  (3498)
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.
EcoO109I  (3496)
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.
AatII  (3442)
1 site
G A C G T C C T G C A G
ZraI  (3440)
1 site
G A C G T C C T G C A G
XmnI  (3119)
1 site
G A A N N N N T T C C T T N N N N A A G
PvuI  (2890)
1 site
C G A T C G G C T A G C
FspI  (2742)
1 site
T G C G C A A C G C G T
AseI  (2692)
1 site
A T T A A T T A A T T A
NmeAIII  (2668)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BglI  (2640)
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.
BsrFI  (2600)
1 site
R C C G G Y Y G G C C R

Cleavage may be enhanced when more than one copy of the BsrFI recognition sequence is present.
After cleavage, BsrFI can remain bound to DNA and alter its electrophoretic mobility.
BsaI  (2581)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
AhdI  (2520)
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.
AlwNI  (2043)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
PspFI  (1935)
1 site
C C C A G C G G G T C G
BseYI  (1931)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
EcoRI  (88)
1 site
G A A T T C C T T A A G
BseRI  (115)
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.
BamHI  (145)
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.
SphI  (155)
1 site
G C A T G C C G T A C G
Eco53kI  (159)
1 site
G A G C T C C T C G A G
BanII  (161)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
SacI  (161)
1 site
G A G C T C C T C G A G
Acc65I  (163)
1 site
G G T A C C C C A T G G
KpnI  (167)
1 site
G G T A C C C C A T G G
TspMI  (168)
1 site
C C C G G G G G G C C C
XmaI  (168)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (170)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SalI  (173)
1 site
G T C G A C C A G C T G
HincII  (175)
1 site
G T Y R A C C A R Y T G
PstI  (183)
1 site
C T G C A G G A C G T C
BsgI  (185)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BfuAI  (186)
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  (186)
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.
BglII  (232)
1 site
A G A T C T T C T A G A
BlpI  (250)
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.
NheI  (358)
1 site
G C T A G C C G A T C G
BmtI  (362)
1 site
G C T A G C C G A T C G
Bpu10I  (383)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
PvuII  (510)
1 site
C A G C T G G T C G A C
BspEI  (606)
1 site
T C C G G A A G G C C T
PasI  (842)
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.
PflMI  (842)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
MscI  (877)
1 site
T G G C C A A C C G G T
BtgI  (911)
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  (911)
1 site
C C A T G G G G T A C C
StyI  (911)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
XbaI  (1214)
1 site
T C T A G A A G A T C T
PfoI  (1271)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
PflFI  (1374)
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  (1374)
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.
BsaAI  (1381)
1 site
Y A C G T R R T G C A Y
BstZ17I  (1400)
1 site
G T A T A C C A T A T G
NdeI  (1450)
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.
BstAPI  (1451)
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.
BspQI  (1511)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (1511)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be mixed before removing an aliquot.
AflIII  (1627)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (1627)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AmpR
2447 .. 3307  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   2447 .. 3238  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2447 .. 3307  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3239 .. 3307  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2447 .. 3307  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
CmR
397 .. 1056  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
397 .. 1056  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
1688 .. 2276  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1688 .. 2276  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ATG
115 .. 117  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
115 .. 117  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
127 .. 144  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
127 .. 144  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
Tag-100
193 .. 228  =  36 bp
12 amino acids  =  1.4 kDa
Product: epitope tag derived from MAP kinase 2
Tag-100
193 .. 228  =  36 bp
12 amino acids  =  1.4 kDa
Product: epitope tag derived from MAP kinase 2
AmpR