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Gene of interest

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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Gene of interest

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  1. 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

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