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).