pHELLSGATE 4

High-throughput binary Gateway® vector for silencing genes in plants using intron-containing hairpin RNA.
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AgeI (17,427) XbaI (17,083) XbaI (15,466) HindIII (15,454) MCS 2 BspDI - ClaI (15,449) PacI (15,338) KpnI (14,653) Acc65I (14,649) MCS 1 EcoRI (14,643) AbsI - XhoI (14,637) 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 4 17,861 bp
AgeI  (17,427)
1 site
A C C G G T T G G C C A
XbaI  (17,083)
2 sites
T C T A G A A G A T C T
XbaI  (15,466)
2 sites
T C T A G A A G A T C T
HindIII  (15,454)
1 site
A A G C T T T T C G A A
BspDI  (15,449)
1 site
A T C G A T T A G C T A
ClaI  (15,449)
1 site
A T C G A T T A G C T A
PacI  (15,338)
1 site
T T A A T T A A A A T T A A T T
KpnI  (14,653)
1 site
G G T A C C C C A T G G
Acc65I  (14,649)
1 site
G G T A C C C C A T G G
EcoRI  (14,643)
1 site
G A A T T C C T T A A G
AbsI  (14,637)
1 site
C C T C G A G G G G A G C T C C
XhoI  (14,637)
2 sites
C T C G A G G A G C T C
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 kanamycin
KanR
448 .. 1269  =  822 bp
273 amino acids  =  29.9 kDa
3 segments
   Segment 3:  nptII  
   499 .. 1269  =  771 bp
   256 amino acids  =  28.1 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
Product: fusion between an N-terminal peptide of nopaline synthase and Tn5 aminoglycoside phosphotransferase
confers resistance to kanamycin
SmR
8600 .. 9388  =  789 bp
262 amino acids  =  29.2 kDa
Product: aminoglycoside adenylyltransferase
confers resistance to spectinomycin and streptomycin
SmR
8600 .. 9388  =  789 bp
262 amino acids  =  29.2 kDa
Product: aminoglycoside adenylyltransferase
confers resistance to spectinomycin and streptomycin
IS1
3015 .. 3782  =  768 bp
prokaryotic transposable element
IS1
3015 .. 3782  =  768 bp
prokaryotic transposable element
PDK intron
14,662 .. 15,428  =  767 bp
pyruvate orthophosphate dikinase intron from Flaveria trinervia
PDK intron
14,662 .. 15,428  =  767 bp
pyruvate orthophosphate dikinase intron from Flaveria trinervia
OCS terminator
17,092 .. 17,798  =  707 bp
octopine synthase terminator
OCS terminator
17,092 .. 17,798  =  707 bp
octopine synthase terminator
oriV
10,071 .. 10,684  =  614 bp
origin of replication for the bacterial F plasmid
oriV
10,071 .. 10,684  =  614 bp
origin of replication for the bacterial F plasmid
ori
6915 .. 7503  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
6915 .. 7503  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CaMV 35S promoter
12,672 .. 13,017  =  346 bp
strong constitutive promoter from cauliflower mosaic virus
CaMV 35S promoter
12,672 .. 13,017  =  346 bp
strong constitutive promoter from cauliflower mosaic virus
ccdB
13,675 .. 13,980  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA gyrase
Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains.
ccdB
13,675 .. 13,980  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA gyrase
Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains.
ccdB
16,025 .. 16,330  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA gyrase
Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains.
ccdB
16,025 .. 16,330  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA gyrase
Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains.
NOS terminator
1894 .. 2146  =  253 bp
nopaline synthase terminator and poly(A) signal
NOS terminator
1894 .. 2146  =  253 bp
nopaline synthase terminator and poly(A) signal
attP1
13,048 .. 13,279  =  232 bp
recombination site for the Gateway® BP reaction
attP1
13,048 .. 13,279  =  232 bp
recombination site for the Gateway® BP reaction
attP2
14,387 .. 14,618  =  232 bp
recombination site for the Gateway® BP reaction (pDONR™221 version)
attP2
14,387 .. 14,618  =  232 bp
recombination site for the Gateway® BP reaction (pDONR™221 version)
attP2
15,490 .. 15,721  =  232 bp
recombination site for the Gateway® BP reaction (pDONR™221 version)
attP2
15,490 .. 15,721  =  232 bp
recombination site for the Gateway® BP reaction (pDONR™221 version)
attP1
16,829 .. 17,060  =  232 bp
recombination site for the Gateway® BP reaction
attP1
16,829 .. 17,060  =  232 bp
recombination site for the Gateway® BP reaction
NOS promoter
264 .. 447  =  184 bp
Function: NOS promoter