The original pHis-parallel1 vector is a gift from S. L. McKnight’s research group at the Department of Biochemistry, University of Texas Southwestern Medical Center. The pHis-parallel1 vector contains two 6× His at both ends of the open reading frame. The TDP43 LC and Mfp5 genes were separately synthesized by GENEWIZ (Suzhou) and then amplified by polymerase chain reaction (PCR) with appropriate overhangs for Gibson assembly. TDP43 LC or recombinant gene TLC-M containing TDP43 LC and Mfp5 was cloned into pHis-parallel1 vectors using one-step isothermal Gibson assembly. The cleaved vector and corresponding PCR products were mixed together with the Gibson Assembly Master 2× Mix (New England BioLabs) at 50°C for 1 hour and then transformed into DH5α Escherichia coli competent cells. PCR amplifications were carried out with primer oligos purchased from GENEWIZ. A Bio-Rad S1000 thermal cycler with dual 48/48 fast reaction modules (Bio-Rad) was used to perform PCR, ligations, and Gibson assembly. Gel extractions were carried out with QIAquick Gel Extraction Kits (QIAGEN). All constructs were sequence-verified by GENEWIZ.

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