Construction of DNA-encoded programs is central to synthetic biology and the chosen method often
determines the time required to design and build constructs for testing. Here we describe and summarise
key features of the available toolkits for DNA construction for mammalian cells. We compare the different
cloning strategies based on their complexity and the time needed to generate constructs of different sizes,
and we reflect on why Golden Gate toolkits now dominate due to their modular design. We look forward to
future advances, including accessory packs to cloning toolkits that can facilitating editing, orthogonality,
advanced regulation, and integration into synthetic chromosome construction.