pET-32 Xa_LIC (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC) to express thioredoxin-tagged proteins with a Factor Xa site.

Sequence Author: MilliporeSigma (Novagen)

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BlpI (5771) DraIII (5449) PsiI (5321) ScaI (4786) PvuI (4676) PstI (4551) BsaI (4367) AhdI (4306) AlwNI (3829) PciI (3413) BspQI - SapI (3297) BstZ17I (3184) PflFI - Tth111I (3158) Bpu10I (2519) 6xHis AvaI - BsoBI - PaeR7I - PspXI - XhoI (5849) EagI - NotI (5857) HindIII (5864) SalI (5870) Eco53kI (5879) SacI (5881) EcoRI (5883) BamHI (5889) EcoRV (5897) NcoI (5902) End (5909) Factor Xa site Start (0) Factor Xa site AgeI (22) Acc65I (25) KpnI (29) BglII (32) BstBI (59) thrombin site 6xHis MscI (142) RsrII (380) RBS XbaI (520) T7 promoter SgrAI (631) SphI (787) EcoNI (847) BstAPI (995) MluI (1312) BclI * (1326) BstEII (1493) PspOMI (1519) ApaI (1523) BssHII (1723) HpaI (1818) PshAI (2157) FspAI (2394) PpuMI (2419) pET-32 Xa/LIC 5909 bp
BlpI  (5771)
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.
DraIII  (5449)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
PsiI  (5321)
1 site
T T A T A A A A T A T T
ScaI  (4786)
1 site
A G T A C T T C A T G A
PvuI  (4676)
1 site
C G A T C G G C T A G C
PstI  (4551)
1 site
C T G C A G G A C G T C
BsaI  (4367)
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  (4306)
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  (3829)
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.
PciI  (3413)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3297)
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  (3297)
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.
BstZ17I  (3184)
1 site
G T A T A C C A T A T G
PflFI  (3158)
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  (3158)
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.
Bpu10I  (2519)
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.
AvaI  (5849)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (5849)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
PaeR7I  (5849)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5849)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5849)
1 site
C T C G A G G A G C T C
EagI  (5857)
1 site
C G G C C G G C C G G C
NotI  (5857)
1 site
G C G G C C G C C G C C G G C G
HindIII  (5864)
1 site
A A G C T T T T C G A A
SalI  (5870)
1 site
G T C G A C C A G C T G
Eco53kI  (5879)
1 site
G A G C T C C T C G A G
SacI  (5881)
1 site
G A G C T C C T C G A G
EcoRI  (5883)
1 site
G A A T T C C T T A A G
BamHI  (5889)
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.
EcoRV  (5897)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
NcoI  (5902)
1 site
C C A T G G G G T A C C
End  (5909)
0 sites
Start  (0)
0 sites
AgeI  (22)
1 site
A C C G G T T G G C C A
Acc65I  (25)
1 site
G G T A C C C C A T G G
KpnI  (29)
1 site
G G T A C C C C A T G G
BglII  (32)
1 site
A G A T C T T C T A G A
BstBI  (59)
1 site
T T C G A A A A G C T T
MscI  (142)
1 site
T G G C C A A C C G G T
RsrII  (380)
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.
XbaI  (520)
1 site
T C T A G A A G A T C T
SgrAI  (631)
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.
SphI  (787)
1 site
G C A T G C C G T A C G
EcoNI  (847)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
BstAPI  (995)
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.
MluI  (1312)
1 site
A C G C G T T G C G C A
BclI  (1326)
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.
BstEII  (1493)
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.
PspOMI  (1519)
1 site
G G G C C C C C C G G G
ApaI  (1523)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BssHII  (1723)
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.
HpaI  (1818)
1 site
G T T A A C C A A T T G
PshAI  (2157)
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.
FspAI  (2394)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (2419)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
lacI
962 .. 2044  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
962 .. 2044  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
AmpR
4233 .. 5093  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   4233 .. 5024  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4233 .. 5093  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   5025 .. 5093  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4233 .. 5093  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3474 .. 4062  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3474 .. 4062  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
5225 .. 5680  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
5225 .. 5680  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
S-Tag
40 .. 84  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
40 .. 84  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
thrombin site
91 .. 108  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
thrombin site
91 .. 108  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
6xHis
118 .. 135  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
118 .. 135  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
TrxA
157 .. 483  =  327 bp
109 amino acids  =  11.8 kDa
Product: E. coli thioredoxin
TrxA
157 .. 483  =  327 bp
109 amino acids  =  11.8 kDa
Product: E. coli thioredoxin
rop
2853 .. 3044  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
rop
2853 .. 3044  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
AmpR promoter
5094 .. 5198  =  105 bp
AmpR promoter
5094 .. 5198  =  105 bp
6xHis
5831 .. 5848  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5831 .. 5848  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
Factor Xa site
5924 .. 5924  =  1 bp
0 codons
Product: Factor Xa recognition and cleavage site
Factor Xa site
5924 .. 5924  =  1 bp
0 codons
Product: Factor Xa recognition and cleavage site
lacI promoter
884 .. 961  =