Resources
Plasmid Files

pHELLSGATE 12

High-throughput binary Gateway® 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) MCS 1 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

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
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

The sticky ends produced by AflII are hard to ligate.
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
   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
   Segment 2:  
   490 .. 498  =  9 bp
   3 amino acids  =  443.6 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
   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 bean catalase intron, modified
NOS promoter
264 .. 447  =  184 bp
nopaline synthase promoter
NOS promoter
264 .. 447  =  184 bp
nopaline synthase promoter
attR1
13,026 .. 13,150  =  125 bp
recombination site for the Gateway® LR reaction
attR1
13,026 .. 13,150  =  125 bp
recombination site for the Gateway® LR reaction
attR2
14,324 .. 14,448  =  125 bp
recombination site for the Gateway® LR reaction
attR2
14,324 .. 14,448  =  125 bp
recombination site for the Gateway® LR reaction
attR2
15,489 .. 15,613  =  125 bp
recombination site for the Gateway® LR reaction
attR2
15,489 .. 15,613  =  125 bp
recombination site for the Gateway® LR reaction
attR1
16,779 .. 16,903  =  125 bp
recombination site for the Gateway® LR reaction
attR1
16,779 .. 16,903  =  125 bp
recombination site for the Gateway® LR reaction
oriT
2846 .. 2955  =  110 bp
incP origin of transfer
oriT
2846 .. 2955  =  110 bp
incP origin of transfer
LB T-DNA repeat
2268 .. 2292  =  25 bp
left border repeat from nopaline C58 T-DNA
LB T-DNA repeat
2268 .. 2292  =  25 bp
left border repeat from nopaline C58 T-DNA
RB T-DNA repeat
11,182 .. 11,206  =  25 bp
right border repeat from nopaline C58 T-DNA
RB T-DNA repeat
11,182 .. 11,206  =  25 bp
right border repeat from nopaline C58 T-DNA
MCS 1
14,456 .. 14,466  =  11 bp
multiple cloning site 1
MCS 1
14,456 .. 14,466  =  11 bp
multiple cloning site 1
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