Hiscribe t7 high yield rna synthesis kit catalog

Picornaviral positive-strand sequences were obtained from NCBI (SVV-001^{45 (link)} (GenBank: DQ641257) and CVA21^{46 (link)} (Genbank: AF546702.1).Custom ribozymes were designed for the 5′ of the viral template, and 30 nucleotide poly adenosine (pA) sequences were added to the 3’ end, followed by either SapI (SVV) or BsmBI (CVA21) restriction site to generate polythymidine templates of the appropriate length after linearization. The SVV-S177A mutation was introduced by point mutagenesis for the SVV-S177A virus utilized in Supplementary Fig. 8, and the Synthetic SVV virus, SVV(S177A-IRES2) is comprised SVV-001 including this mutation and an improved IRES, SVV IRES2. This sequence is derived from SVA/Canada/MB/NCFAD-104-1/2015 (GenBank: KY486156). The IVT templates were constructed with synthetic dsDNA fragments (IDT Geneblocks, Genscript, Piscataway, NJ) and Gibson assembly (NEBuilder HiFi DNA Assembly Master Mix, Catalog #E2621L, NEB, Ipswich, MA) following the manufacturer protocol. These constructs were sequenced end to end, linearized with either SapI (SVV) or BsmBI (CVA21) (NEB SapI # R0569L, BsmBI # R0739L, Ipswich, MA), and research-grade IVTs (NEB HiScribe T7 High Yield RNA Synthesis Kit Catalog #E2040S, Ipswich, MA) were performed to ensure viral kickoff. All viral stocks used in this work were obtained with these reverse genetics systems.