pLATE52 (linearized)

Vector for ligation independent cloning (LIC) and tightly regulated bacterial expression of a protein with a WELQut-cleavable N-terminal 6xHis tag.

Sequence Author: Thermo Fisher

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lac operator LIC reverse sequencing primer (4297 .. 4312) rrnB T2 terminator rrnB T1 terminator AatII (4004) ZraI (4002) SspI (3886) ScaI (3562) PvuI (3452) FspI (3304) BglI (3202) AhdI (3082) AlwNI (2605) PciI (2189) BspQI - SapI (2073) NdeI (2012) BstZ17I (1962) AccI (1961) BsaAI (1943) PflFI - Tth111I (1936) PfoI (1833) AfeI (1445) BsaBI * (1388) BspEI * (1380) PluTI (1289) Eco53kI (4315) SacI (4317) Acc65I (4319) KpnI (4323) MauBI (4330) BfuAI - BspMI - PaqCI (4335) SbfI (4346) AscI (4351) LIC forward sequencing primer (4360 .. 4379) T7 promoter LIC reverse sequencing primer (4387 .. 4401) lac operator XbaI (4407) SwaI (4432) RBS ATG NheI (4491) BmtI (4495) T7 tag (gene 10 leader) End (4526) Start (0) PmeI (18) StuI (25) SphI (33) PacI (55) LIC reverse sequencing primer (62 .. 85) HindIII (123) BspDI - ClaI (130) BamHI (157) StyI (173) T7 terminator PflMI (228) MluI (646) BclI * (660) BstEII (827) PspOMI (853) ApaI (857) ApoI (921) EcoRV (1096) HpaI (1152) KasI (1285) NarI * (1286) SfoI (1287) pLATE52 4526 bp
AatII  (4004)
1 site
G A C G T C C T G C A G
ZraI  (4002)
1 site
G A C G T C C T G C A G
SspI  (3886)
1 site
A A T A T T T T A T A A
ScaI  (3562)
1 site
A G T A C T T C A T G A
PvuI  (3452)
1 site
C G A T C G G C T A G C
FspI  (3304)
1 site
T G C G C A A C G C G T
BglI  (3202)
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.
AhdI  (3082)
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  (2605)
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  (2189)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2073)
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  (2073)
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.
NdeI  (2012)
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.
BstZ17I  (1962)
1 site
G T A T A C C A T A T G
AccI  (1961)
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.
BsaAI  (1943)
1 site
Y A C G T R R T G C A Y
PflFI  (1936)
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  (1936)
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.
PfoI  (1833)
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.
AfeI  (1445)
1 site
A G C G C T T C G C G A
BsaBI  (1388)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
BspEI  (1380)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
PluTI  (1289)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
Eco53kI  (4315)
1 site
G A G C T C C T C G A G
SacI  (4317)
1 site
G A G C T C C T C G A G
Acc65I  (4319)
1 site
G G T A C C C C A T G G
KpnI  (4323)
1 site
G G T A C C C C A T G G
MauBI  (4330)
1 site
C G C G C G C G G C G C G C G C
BfuAI  (4335)
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  (4335)
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.
PaqCI  (4335)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the PaqCI recognition sequence.
Sticky ends from different PaqCI sites may not be compatible.
Cleavage can be improved with PaqCI Activator.
SbfI  (4346)
1 site
C C T G C A G G G G A C G T C C
AscI  (4351)
1 site
G G C G C G C C C C G C G C G G
XbaI  (4407)
1 site
T C T A G A A G A T C T
SwaI  (4432)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
NheI  (4491)
1 site
G C T A