Cloning Should Be Simple: <i>Escherichia coli</i> DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

<div><p>Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex <i>in vitro</i> or <i>in vivo</i> assembly reactions followed by plasmid propagation in recombination-impaired <i>Escherichia coli</i> strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids <i>in vivo</i>. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of <i>E</i>. <i>coli</i> may enable productivity gain across disciplines involving recombinant DNA work.</p>