The bacterial strains and plasmids used in this study are described in Table 2. Oligonucleotides were purchased from Integrated DNA Technologies and are listed in Table 3. Genomic DNA was isolated using the Qiagen 100/G genomic tip (Qiagen). Weakening of the staphylococcal cell wall required the addition of 100 µg of lysostaphin (Ambi) into the lysis buffer and incubation at 37°C for 30 min. Plasmids and PCR products were isolated using the Wizard plus kits (Promega), with T4 DNA ligase also purchased from Promega. Plasmids were isolated from staphylococci as described previously (3 (link)). Restriction enzymes, T4 DNA polymerase, and Phusion DNA polymerase were purchased from New England Biolabs. Phire Hotstart DNA polymerase was purchased from Thermofisher. Sanger sequencing was supplied by Eurofins. Routine manipulation of S. aureus and E. coli was performed as described by Monk et al. (3 (link)). X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; Melford) was used at 50 µg/ml in E. coli and 100 µg/ml in S. aureus. Antibiotics were purchased from Sigma Aldrich and used at the following concentrations: carbenicillin (Car), 100 µg/ml; chloramphenicol (Cm), 10 µg/ml; and kanamycin (Kan), 50 µg/ml (E. coli) and 100 µg/ml (S. aureus).