Pmod b2303

The 35S:GFP and CmYLCV:GFP vectors were used for checking the transformation efficiency in this study. Three intermediate module plasmids A, B, and C were prepared for the construction of the CRISPR–Cas9 vector of Ara h 2 [46 (link)]. For module A, CmYLCV promoter from pMOD_A3003 (Addgene #91043) was inserted into pMOD_A0101 (Addgene #90998) in place of 35S promoter via restriction digestion and cloned using T4 Ligase (NEB, Ipswich, MA, USA) (Figure S2A,B). The pMOD_B2303 vector was used for module B. The polycistronic tRNA–gRNA (PTG) gene containing two sgRNAs sequences for Ara h 2 [47 (link)] was synthesized and incorporated commercially into pUC57 (Genscript Biotech Ltd., Piscataway, NJ, USA). The synthesized pUC57-PTG was digested with PstI and XhoI and cloned into the PstI and XhoI-digested pMOD_B2303 vector (Addgene #91068) using T4 Ligase (NEB) following the manufacturer’s recommendations (Figure S2A,C). Modified pMOD_A0101, modified pMOD_B2303, and empty vector pMOD_C0000 (Addgene #91081) were assembled into a non-binary vector, pTRANS_100 (Addgene #91198) by simple Golden Gate protocol using the AarI enzyme [47 (link)] (Figure S2A,D).