pHELLSGATE 12

High-throughput binary Gateway® destination vector with two introns, for silencing genes in plants using intron-containing hairpin RNA.
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AgeI (17,247) XbaI (16,903) XbaI (15,484) BspDI - ClaI (15,262) PacI (15,151) PDK intron KpnI (14,465) Acc65I (14,461) EcoRI (14,455) XhoI (14,449) XhoI (13,020) ScaI (12,386) StuI (12,234) SacI (11,646) Eco53kI (11,644) PmeI (11,220) BsiWI (11,055) FseI (10,595) Bsu36I (330) RsrII (1119) AflII (1923) MauBI (2118) LB T-DNA repeat BspEI (3460) AleI (5445) SexAI * (5533) FspAI (6101) NruI (6598) pHELLSGATE 12 17,681 bp
AgeI  (17,247)
1 site
A C C G G T T G G C C A
XbaI  (16,903)
2 sites
T C T A G A A G A T C T
XbaI  (15,484)
2 sites
T C T A G A A G A T C T
BspDI  (15,262)
1 site
A T C G A T T A G C T A
ClaI  (15,262)
1 site
A T C G A T T A G C T A
PacI  (15,151)
1 site
T T A A T T A A A A T T A A T T
KpnI  (14,465)
1 site
G G T A C C C C A T G G
Acc65I  (14,461)
1 site
G G T A C C C C A T G G
EcoRI  (14,455)
1 site
G A A T T C C T T A A G
XhoI  (14,449)
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
14,475 .. 15,241  =  767 bp
pyruvate orthophosphate dikinase intron from Flaveria trinervia
PDK intron
14,475 .. 15,241  =  767 bp
pyruvate orthophosphate dikinase intron from Flaveria trinervia
OCS terminator
16,912 .. 17,618  =  707 bp
octopine synthase terminator
OCS terminator
16,912 .. 17,618  =  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
13,579 .. 13,884  =  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
13,579 .. 13,884  =  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
15,942 .. 16,247  =  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
15,942 .. 16,247  =  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
cat1 intron
15,276 .. 15,465  =  190 bp
castor bean catalase intron, modified
cat1 intron
15,276 .. 15,465  =  190 bp
castor bea