The pVP26-Flag, with a Flag tag fused to the carboxyl-terminus of the VP26 gene, was constructed using pE1 as the backbone vector [31] via a two-step Red (en passant) recombination strategy [63,64] (Figure 9). The PCR fragment P1 carrying homo-arm1 (homologous to the upstream sequence of the UL35 stop codon)-hom B-I-SceI-kan-hom B-Flag tag-homo-arm2 (homologous to the UL35 stop codon and downstream sequence) was synthesized by Tsingke Biotech. Co., Ltd. (Beijing, China) and used to generate pVP26-Flag. pVP26-Flag was confirmed via sequencing.
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