We used the penton base protein from human Ad3 (GenBank Z29487) as a starting point and implemented a BioBrick format (22) to functionalize one locus within the VL and two loci in the RGD loop for facile and rapid custom DNA insertion (fig. S1). The insertion loci were flanked by unique restriction sites that were eliminated from elsewhere in the Ad3 penton base encoding gene. To preserve structural integrity of the penton base protomer, the restriction sites were designed in silico to be located outside of and adjacent to known secondary structure elements in the penton base protein. The resulting gene encoding for ADDomer was synthesized, codon-optimized for recombinant expression, and pasted into plasmid pACEBac1 (Geneva Biotech SARL, Switzerland) (26) using Bam HI and Hind III restriction sites, giving rise to pACEBac-ADDomer. The primary sequence of the encoded ADDomer is provided in fig. S2. In this study, pACEBac-ADDomer served as the backbone for inserting DNA encoding for selected epitopes into the designed insertion loci in the variable and RGD loops. Primary sequences of the inserted epitopes in this study are listed in table S2.

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