Generating IVT-produced mRNA using DNA plasmids

Construction of DNA plasmids to generate in vitro transcription (IVT)-produced mRNA was performed as previously described^{62 (link)}. Briefly, DNA constructs containing genes of interest were ordered from Dharmacon or Addgene. Primers were designed that spanned the open reading frame (ORF) of the genes of interest. PCR reactions were performed using HiFi HotStart DNA polymerase (KAPA Biosystems) per the manufacturer’s instructions. Amplified ORFs were treated using T4 polynucleotide kinase (New England Biolabs). PCR products were then purified using QIAquick spin columns (Qiagen). Blunt ligations between the PCR products and a template plasmid (pORF-in) that incorporates generic 5’ and 3’ untranslated regions (UTRs) and a poly-A tail, as previously described^{62 (link)}, were performed using T4 ligase (New England Biolabs). Newly ligated plasmids were propagated using the TOPO TA cloning kit (ThermoFisher Scientific) and verified by Sanger sequencing in the KI gene facility (Stockholm, Sweden).