FIG 1.

Generation of pKW-L2ori. (A) Species-specific iterons from C. trachomatis pL2 and C. muridarum pNigg showing 4 (I to IV) 22-bp tandem repeats (yellow) and immediate flanking DNA (blue). (B) Schematic of the pKW-L2ori shuttle vector, constructed as a backbone for downstream gene deletion applications. An aadA-gfp cassette is flanked by restriction sites (SbfI/SalI) for the subsequent introduction of ~3 kb of genomic sequences flanking the target gene. The constitutively expressed mCherry gene is included as a fluorescence marker of vector maintenance. The 265-bp region containing the pL2 iteron represents the putative chlamydial plasmid ori, and pBR322 is present to enable cloning manipulations in E. coli. LoxP sites were included to enable the subsequent CRE-mediated excision of the integrated aadA-gfp cassette from the chlamydial genome.