pHELLSGATE 8

High-throughput binary Gateway® vector for silencing genes in plants using intron-containing hairpin RNA.
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AgeI (17,051) XbaI (16,707) XbaI (15,280) HindIII (15,268) BspDI - ClaI (15,263) PacI (15,152) KpnI (14,466) Acc65I (14,462) MCS 1 EcoRI (14,456) XhoI (14,450) PspXI - XhoI (13,020) ScaI (12,386) StuI (12,234) SacI (11,646) Eco53kI (11,644) PmeI (11,220) BsiWI (11,055) FseI (10,595) OCS terminator Bsu36I (330) RsrII (1119) AflII (1923) MauBI (2118) LB T-DNA repeat BspEI (3460) AleI (5445) SexAI * (5533) FspAI (6101) NruI (6598) pHELLSGATE 8 17,485 bp
AgeI  (17,051)
1 site
A C C G G T T G G C C A
XbaI  (16,707)
2 sites
T C T A G A A G A T C T
XbaI  (15,280)
2 sites
T C T A G A A G A T C T
HindIII  (15,268)
1 site
A A G C T T T T C G A A
BspDI  (15,263)
1 site
A T C G A T T A G C T A
ClaI  (15,263)
1 site
A T C G A T T A G C T A
PacI  (15,152)
1 site
T T A A T T A A A A T T A A T T
KpnI  (14,466)
1 site
G G T A C C C C A T G G
Acc65I  (14,462)
1 site
G G T A C C C C A T G G
EcoRI  (14,456)
1 site
G A A T T C C T T A A G
XhoI  (14,450)
2 sites
C T C G A G G A G C T C
PspXI  (13,020)
1 site
V C T C G A G B B G A G C T C V
XhoI  (13,020)
2 sites
C T C G A G G A G C T C
ScaI  (12,386)
1 site
A G T A C T T C A T G A
StuI  (12,234)
1 site
A G G C C T T C C G G A
SacI  (11,646)
1 site
G A G C T C C T C G A G
Eco53kI  (11,644)
1 site
G A G C T C C T C G A G
PmeI  (11,220)
1 site
G T T T A A A C C A A A T T T G
BsiWI  (11,055)
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.
FseI  (10,595)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
Bsu36I  (330)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
RsrII  (1119)
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.
AflII  (1923)
1 site
C T T A A G G A A T T C
MauBI  (2118)
1 site
C G C G C G C G G C G C G C G C
BspEI  (3460)
1 site
T C C G G A A G G C C T
AleI  (5445)
1 site
C A C N N N N G T G G T G N N N N C A C
SexAI  (5533)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
FspAI  (6101)
1 site
R T G C G C A Y Y A C G C G T R
NruI  (6598)
1 site
T C G C G A A G C G C T
trfA
4407 .. 5555  =  1149 bp
382 amino acids  =  43.8 kDa
Product: trans-acting replication protein that binds to and activates oriV
trfA
4407 .. 5555  =  1149 bp
382 amino acids  =  43.8 kDa
Product: trans-acting replication protein that binds to and activates oriV
KanR
448 .. 1269  =  822 bp
273 amino acids  =  29.9 kDa
3 segments
   Segment 1:  nos  
   448 .. 489  =  42 bp
   14 amino acids  =  1.4 kDa
Product: fusion between an N-terminal peptide of nopaline synthase and Tn5 aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
448 .. 1269  =  822 bp
273 amino acids  =  29.9 kDa
3 segments
   Segment 2:  
   490 .. 498  =  9 bp
   3 amino acids  =  443.5 Da
Product: fusion between an N-terminal peptide of nopaline synthase and Tn5 aminoglycoside phosphotransferase
confers resistance to kana