A 355 bp fragment containing the 89K oriT sequence was amplified from the 05ZYH33 chromosome using PCR primers oriT-F and oriT-R. The resulting PCR products were cloned into pMD18-T, creating pMD-oriT. Plasmid pMD-oriT was digested with EcoRI and BamHI to release the oriT fragment, which was then cloned into the E. coli-S. suis pVA838 shuttle vector, generating pVA838-oriT. Plasmids pET-MobA89K and pET-MobC89K were created by inserting the mobA89K and mobC89K PCR products (amplified with specific primer pairs mobA-F/mobA-R and mobC-F/mobC-R respectively) into the NdeI/XhoI sites of the pET-21a(+) expression vector, creating fusion proteins with a C-terminal His6 tag. A 774 bp mobA89KN258 fragment encoding the first 258 aa of MobA89K was also amplified using primers mobA-F/N258-R and cloned into the unique NdeI and XhoI sites of pET-28a(+), creating a fusion protein with His6 tags at both its Nand C-termini. All constructs were verified by DNA sequence analysis.