*3.1. Construction of Chimeric Insertion*

The coding sequence of phospholipase PldA *Y. pseudotuberculosis* strain 488 (Gen-Bank: MW848438) without signal sequence was amplified with primers: PldA-NdeI 5- -CCG**CATATG**GAAGCAACGATTGAAAAGATTC-3- (forward) and PldA-Linker-R 5- gttaattaaaccagcaccgtcaccAAGGACATCGTTCAACATGATAC-3- (reverse). The pTurboGFP plasmid (Evrogen, Russia; GenBank: ASW25889.1) was used as a template for *gfp* amplification with primers: GFP-linker-F 5- -ggtgacggtgctggtttaattaacGCAGAAATCTATAACAAA GATGG-3- (forward) and GFP-R 5- -TGAT**CTCGAG**TTATTCTTCACCGGCATCTGCATC CG-3- (reverse). Bases in uppercase letters indicate *pldA* and *gfp* specific regions. Two restriction sites, *NdeI* and *XhoI*, (in bold letters) were incorporated at the 5 and 3 ends of the resulting insertion. In order to connect and improve the assembly of *pldA* and *gfp* we added the sequence coding flexible linker (GDGAGLIN) to the *pldA* reverse primer and the *gfp* forward primer (in lowercase letters). The gene fusion was assembled by II-rounded asymmetric PCR. At the first round, the *pldA* and *gfp* fragments were amplified in total volume of 20 μLeach. Reaction mixes included 250 μM of each dNTP, 0.2 U of Q5 High-Fidelity DNA Polymerase (Promega, Madison, WI, USA), 5× Q5 Reaction Buffer, 0.05 μM forward primer, and 0.5 μM reverse primer (for *pldA* production), or 0.5 μM forward primer and 0.05 μM reverse primer (for *gfp* production). The PCR conditions were as follows: initial denaturation at 95 ◦C for 5 min, then 21 cycles of 94 ◦C for 30 s, 55 ◦C for 30 s, and 72 ◦C for 40 s, followed by a final extension step at 72 ◦C for 2 min. In the second round, the reaction products were mixed, then an additional 0.2 U of Q5 High-Fidelity DNA Polymerase was added, and the combined reaction mix was amplified for 15 cycles at 94 ◦C for 30 s, 55 ◦C for 30 s, and 72 ◦C for 1 min, followed by a final extension step at 72 ◦C for 5 min. After amplification, the PCR fragment corresponding to the expected length of the chimeric insertion was purified and used for cloning.
