Primer designation
All oligos were synthesized in Sangon Biotech (China) and listed in Table 2.
High-fidelity polymerase PrimeSTAR® GXL DNA polymerase (Takara, Japan) were used for amplifying linear dsDNA targeting fragments. Primers used to produce these targeting fragments were chimeric primers containing two parts: 38 bases homologous to the sequence surrounding the target locus were designed at 5’ end of primers, while about 20 bases of amplifying sequence were at 3’ end. After purification following PCR amplification, linear dsDNA was electroporated into cells for Red recombination. Single-stranded DNA, sspigA-F, used to deletePBAD-E-GmRor PBAD-kil-sd-E-GmRselection/counter-selection cassettes inserted into the pigAlocus of S. marcescens was reported in our previous study (W. Chen, Chen, & Cao, 2021).