Table 2.

Selection rules for barcode insertion points

Rule	Purpose
Target region must be located in the chromosome	Maximize stability by incorporation on major replicon
Insertion point must lie near the midpoint of an intergenic space larger than 500 bp	Minimize disruption of potential coding sequences or regulatory elements
No annotated genes or potential ORFs in the intergenic space	Minimize disruption of potential coding sequences or regulatory elements
Must lie between two convergently transcribed genes	Minimize disruption of potential coding sequences or regulatory elements
No repetitive structure in intergenic space	Facilitate synthesis and cloning of constructs and minimize potential issues with homologous recombination
No identical repetitive elements >200 bp in size within 10,000 bp	Minimize 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 draft	Maximize likelihood of success in selected target strain
Target must be present in commercial B. thuringiensis subsp. kurstaki isolate	Maximize versatility and adaptability of barcode targeting vectors to different strains