title: GreenGate---a novel, versatile, and efficient cloning system for plant transgenesis.
abstract: Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project. Several alternative cloning systems have been developed in recent years to overcome these issues, including the type IIS enzyme based Golden Gate technique. Here we introduce our GreenGate system for rapidly assembling plant transformation constructs, which is based on the Golden Gate method. GreenGate cloning is simple and efficient since it uses only one type IIS restriction endonuclease, depends on only six types of insert modules (plant promoter, N-terminal tag, coding sequence, C-terminal tag, plant terminator and plant resistance cassette), but at the same time allows assembling several expression cassettes in one binary destination vector from a collection of pre-cloned building blocks. The system is cheap and reliable and when combined with a library of modules considerably speeds up cloning and transgene stacking for plant transformation.
authors.affiliation: Centre for Organismal Studies, Heidelberg University, Heidelberg, Baden-Württemberg, Germany.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Lampropoulos
authors.affiliation: Centre for Organismal Studies, Heidelberg University, Heidelberg, Baden-Württemberg, Germany.
authors.forename: Zoran
authors.initials: Z
authors.lastname: Sutikovic
authors.affiliation: Centre for Organismal Studies, Heidelberg University, Heidelberg, Baden-Württemberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Wenzl
authors.affiliation: Centre for Organismal Studies, Heidelberg University, Heidelberg, Baden-Württemberg, Germany.
authors.forename: Ira
authors.initials: I
authors.lastname: Maegele
authors.affiliation: Centre for Organismal Studies, Heidelberg University, Heidelberg, Baden-Württemberg, Germany.
authors.forename: Jan U
authors.initials: JU
authors.lastname: Lohmann
authors.affiliation: Centre for Organismal Studies, Heidelberg University, Heidelberg, Baden-Württemberg, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Forner
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D013728:Terminator Regions, Genetic; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D010940:Plant Proteins; D015252:Deoxyribonucleases, Type II Site-Specific