title: Rapid hierarchical assembly of medium-size DNA cassettes.
abstract: Synthetic biology applications call for efficient methods to generate large gene cassettes that encode complex gene circuits in order to avoid simultaneous delivery of multiple plasmids encoding individual genes. Multiple methods have been proposed to achieve this goal. Here, we describe a novel protocol that allows one-step cloning of up to four gene-size DNA fragments, followed by a second assembly of these concatenated sequences into large circular DNA. The protocols described here comprise a simple, cheap and fast solution for routine construction of cassettes with up to 10 gene-size components.
authors.affiliation: FAS Center for Systems Biology, Harvard University, 52 Oxford St, Cambridge, MA 02138, USA.
authors.forename: Jonathan Leo
authors.initials: JL
authors.lastname: Schmid-Burgk
authors.forename: Zhen
authors.initials: Z
authors.lastname: Xie
authors.forename: Stefan
authors.initials: S
authors.lastname: Frank
authors.forename: Sebastian
authors.initials: S
authors.lastname: Virreira Winter
authors.forename: Sibylle
authors.initials: S
authors.lastname: Mitschka
authors.forename: Waldemar
authors.initials: W
authors.lastname: Kolanus
authors.forename: Andrew
authors.initials: A
authors.lastname: Murray
authors.forename: Yaakov
authors.initials: Y
authors.lastname: Benenson
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase; D005796:Genes; D016133:Polymerase Chain Reaction
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase