Table 2

Selection rules for barcode insertion points

RulePurpose
Target region must be located in the chromosomeMaximize stability by incorporation on major replicon
Insertion point must lie near the midpoint of an intergenic space larger than 500 bpMinimize disruption of potential coding sequences or regulatory elements
No annotated genes or potential ORFs in the intergenic spaceMinimize disruption of potential coding sequences or regulatory elements
Must lie between two convergently transcribed genesMinimize disruption of potential coding sequences or regulatory elements
No repetitive structure in intergenic spaceFacilitate synthesis and cloning of constructs and minimize potential issues with homologous recombination
No identical repetitive elements >200 bp in size within 10,000 bpMinimize potential loss by deletion via homologous recombination between repeat elements (e.g., insertion sequences)
Target must be intact and consistently annotated in two or more available B. thuringiensis subsp. kurstaki sequences and in ATCC 33679 draftMaximize likelihood of success in selected target strain
Target must be present in commercial B. thuringiensis subsp. kurstaki isolateMaximize versatility and adaptability of barcode targeting vectors to different strains