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Gene of interest. Unique Sequence. Sense Oligo. Sense Oligo. Target sequence (Antisense). Target sequence (Antisense). Target sequence (Sense). PacI. NotI. PacI. 9bp loop. NotI. GCGGCCGC. 3’. TTAATTAA. 5’. 5’. TTAATTAA. TTCAAGAGA. GCGGCCGC. 3’. 45 bp. 45 bp. 90 bp. 3’.

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slide1

Gene of interest

Unique Sequence

Sense Oligo

Sense Oligo

Target sequence

(Antisense)

Target sequence

(Antisense)

Target sequence (Sense)

PacI

NotI

PacI

9bp loop

NotI

GCGGCCGC

3’

TTAATTAA

5’

5’

TTAATTAA

TTCAAGAGA

GCGGCCGC

3’

45 bp

45 bp

90 bp

3’

AATTAATT

CGCCGGCG

5’

3’

5’

AATTAATT

AAGTTCTCT

CGCCGGCG

Complimentary Oligo

Complimentary Oligo

Mix 10 µg of each oligo and heat at 65°C for 20 minutes and incubate at 22°C for 1 hour

Digested overnight with 20 units each of Pac1 and Not1 enzyme

GC

5’

TTCAAGAGA

GC

5’

TAA

3’

3’

TAA

3’

CGCCGG

3’

AAGTTCTCT

TAATT

5’

TAATT

CGCCGG

5’

Ligate into Pac1 and Not1 restricted γ genome of BSMV-VIGS vector

Hairpin-encoding VIGS construct

Antisense VIGS construct

Figure S2