Abstract
The AdenoBuilder platform enables the in vitro assembly of recombinant vectors from plasmid inserts that span the adenovirus genome. Two advantages of AdenoBuilder are the ease of modifying the genome and the ability to produce multicomponent vectors in a single step, facilitating parallel approaches to vector optimization. This protocol describes how to introduce transgenes in place of the endogenous Human Adenovirus serotype 5 (HAd5) E1 and/or E3 genes and can be applied to other parts of the HAd5 genome. For complete details on the use and execution of this protocol, please refer to Miciak et al. (2018).