VPS13D DNA plasmid generation v1

This protocol describes the basic molecular cloning technique utilized for the generation of VPS13D constructs in https://doi.org/10.1083/jcb.202010004. This protocol and the enzymes included in it are commercialized by Takara Bio. Due to low expression of big DNA constructs (>10kb), we decided to generate a codon-optimized cDNA encoding for human VPS13D, including an mScarlet fluorescent protein after aminoacid 1576 flanked by BamHI restriction enzyme sites. The construct was generated by and purchased from Genscript. From this initial construct, we generated several VPS13D constructs. The specific primers and enzymes used for each construct are included in Table 1 of our manuscript: https://doi.org/10.1083/jcb.202010004" . For most of our cloning procedures, Infusion cloning (Takara) was used.