*2.1. Construction of the BCG.HTI2auxo.int Strain Using an Antibiotic-Free Plasmid Selection System*

The double auxotrophic *E. coli*–mycobacterial shuttle integrative vector, the p2auxo.int plasmid, was previously constructed in our laboratory [33]. This vector contains the *glyA* and *LysA* genes, which function as an antibiotic-free selection and maintenance system in the auxotrophic strains of *E. coli* M15Δ*glyA* and BCGΔ*Lys*, respectively. It also contains sites (*attP*) for integration into the BCG genome at the *attB* site. The synthetic sequence of HTI [7] was codon-optimized for BCG expression to match the G+C rich mycobacterial codon usage for enhanced expression [34]. The HTI G+C rich DNA sequence was synthesized by Geneart (USA) and ligated to the integrative p2auxo.int plasmid fused to the 19-kDa lipoprotein secretion signal sequence generating p2auxo.HTIint. The ligation products were subsequently transformed into the *E. coli* M15Δ*glyA* strain for growth and selection.
