Circ-SMO overexpression plasmid, circ-SMO-3xFlag plasmid, 193a.a.-3xFlag plasmid, mutSD plasmid, circ-frame Del plasmid, circ-SMO noATG plasmid, and circ-SMO Mut plasmid were generated by chemical gene synthesis, and pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector was used as plasmid backbone. For validation of IRES activity, the EMCV-IRES sequence, potential circ-SMO IRES sequence (367-515 bp), and IERS-Delete sequences were chemical synthesized and inserted into the middle of “uc” and “RL” sequences using Circ-RLuc-IRES-Reporter vector. 8xGliBS-Luc plasmid was generated by inserting eight tandem copies of the Gli-binding element (5′-GACCACCCA-3′) into the upstream region of minimal promoter TA and the luciferase gene. For FUS promoter luciferase reporter plasmid construction, the wildtype and mutant FUS promoter region were chemically synthesized and inserted into pGL3-Basic-Luciferase vector (Promega, Madison, WI, USA). SMO-HA, N-HA, TMs-HA, and C-HA vectors use pCDNA3.1(+) plasmid backbone. Plasmids above were obtained from Shanghai Generay Biotech Co, Ltd. (Shanghai, China). PTCH1 and Gli1 overexpression plasmid were obtained from Vigene bioscience, Inc. (Jinan, Shandong, China). The plasmids were transfected with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

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