pEarleyGate 202

Gateway®-compatible plant transformation vector with an N-terminal FLAG tag.

Sequence Author: Pikaard Lab

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LB T-DNA repeat BclI * (11,539) PsiI (11,408) BspHI (11,082) EcoRV (10,659) PluTI (9400) SfoI (9398) NarI (9397) KasI (9396) EcoNI (8924) BmtI (8615) NheI (8611) BsiWI (8495) PmeI (5483) AseI (92) Acc65I (407) KpnI (411) FspAI (521) PaqCI (585) AfeI (758) BstBI (1770) StuI (1868) PshAI (2467) PaeR7I - PspXI - XhoI (2654) TMV Ω ATG FLAG attR1 lac UV5 promoter BspEI (3209) BbvCI (3938) SrfI (4084) BstXI (4201) AvrII (4473) PacI (4519) SpeI (4531) MfeI (4835) BstEII (5107) AhdI (5237) SbfI (5262) HindIII (5270) PvuI (5396) pEarleyGate 202 11,776 bp
BclI  (11,539)
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.
PsiI  (11,408)
1 site
T T A T A A A A T A T T
BspHI  (11,082)
1 site
T C A T G A A G T A C T
EcoRV  (10,659)
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.
PluTI  (9400)
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.
SfoI  (9398)
1 site
G G C G C C C C G C G G
NarI  (9397)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI recognition sequence.
KasI  (9396)
1 site
G G C G C C C C G C G G
EcoNI  (8924)
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.
BmtI  (8615)
1 site
G C T A G C C G A T C G
NheI  (8611)
1 site
G C T A G C C G A T C G
BsiWI  (8495)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
PmeI  (5483)
1 site
G T T T A A A C C A A A T T T G
AseI  (92)
1 site
A T T A A T T A A T T A
Acc65I  (407)
1 site
G G T A C C C C A T G G
KpnI  (411)
1 site
G G T A C C C C A T G G
FspAI  (521)
1 site
R T G C G C A Y Y A C G C G T R
PaqCI  (585)
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.
AfeI  (758)
1 site
A G C G C T T C G C G A
BstBI  (1770)
1 site
T T C G A A A A G C T T
StuI  (1868)
1 site
A G G C C T T C C G G A
PshAI  (2467)
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.
PaeR7I  (2654)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2654)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2654)
1 site
C T C G A G G A G C T C
BspEI  (3209)
1 site
T C C G G A A G G C C T
BbvCI  (3938)
1 site
C C T C A G C G G A G T C G
SrfI  (4084)
1 site
G C C C G G G C C G G G C C C G
BstXI  (4201)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
AvrII  (4473)
1 site
C C T A G G G G A T C C
PacI  (4519)
1 site
T T A A T T A A A A T T A A T T
SpeI  (4531)
1 site
A C T A G T T G A T C A
MfeI  (4835)
1 site
C A A T T G G T T A A C
BstEII  (5107)
1 site
G G T N A C C C C A N </