Extended Data Fig. 3: Deletion of putative cryptic toxic ORF in MBaMV L gene.
From: Metagenomics-enabled reverse-genetics assembly and characterization of myotis bat morbillivirus
The first 1,500 nt of the L gene was refractory to cloning. Inspection of the L gene sequence revealed a putative ORF of 86 aa in the complementary (-) strand of the first 1,500 nt of the L gene (a, original sequence and b, ORF-X). To determine if this putative ORF might be toxic to the bacteria used for cloning the MBaMV genome, we made two mutations that were silent in the L ORF but which abolished the initiator methionine(s) in this putative ORF-X. Red shaded nucleotides correspond to third codon of methionine in ORF-X (a, Deleted ORF-X). This “ORF-X KO” MBaMV genomic clone was the eventual construct that was rescued. The amino acid sequence of ORF-X is shown in (b).