title: HomeRun Vector Assembly System: a flexible and standardized cloning system for assembly of multi-modular DNA constructs.
abstract: Advances in molecular and synthetic biology call for efficient assembly of multi-modular DNA constructs. We hereby present a novel modular cloning method that obviates the need for restriction endonucleases and significantly improves the efficiency in the design and construction of complex DNA molecules by standardizing all DNA elements and cloning reactions. Our system, named HomeRun Vector Assembly System (HVAS), employs a three-tiered vector series that utilizes both multisite gateway cloning and homing endonucleases, with the former building individual functional modules and the latter linking modules into the final construct. As a proof-of-principle, we first built a two-module construct that supported doxycycline-induced expression of green fluorescent protein (GFP). Further, with a three-module construct we showed quantitatively that there was minimal promoter leakage between neighbouring modules. Finally, we developed a method, in vitro Cre recombinase-mediated cassette exchange (RMCE) cloning, to regenerate a gateway destination vector from a previous multisite gateway cloning reaction, allowing access to existing DNA element libraries in conventional gateway entry clones, and simple creation of constructs ready for in vivo RMCE. We believe these methods constitute a useful addition to the standard molecular cloning techniques that could potentially support industrial scale synthesis of DNA constructs.
authors: Division of Endocrinology and Diabetes, Department of Medicine, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors: Ming V
authors: MV
authors: Li
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors: Dip
authors: D
authors: Shukla
authors: Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota, United States of America.
authors: Brian H
authors: BH
authors: Rhodes
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors: Anjali
authors: A
authors: Lall
authors: Division of Endocrinology and Diabetes, Department of Medicine, University of Minnesota, Minneapolis, Minnesota, United States of America; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America; Center for Genome Engineering and Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors: Jingmin
authors: J
authors: Shu
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America; Center for Genome Engineering and Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors: Branden S
authors: BS
authors: Moriarity
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America; Center for Genome Engineering and Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors: David A
authors: DA
authors: Largaespada
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D005822:Genetic Vectors; D045325:HCT116 Cells; D006801:Humans
chemical_list: D004247:DNA