The cloning of plasmids can be time-consuming or expensive. Yet, cloning is a prerequisite for many standard experiments for the functional analysis of genes, including the generation of deletion mutants and the localization of gene products. Here, we provide Golden Gate vectors for fast and easy cloning of gene fusion as well as gene deletion vectors applicable to diverse fungi. In Golden Gate cloning, restriction and ligation occur simultaneously in a one-pot reaction. Our vector set contains recognition sites for the commonly used type IIS restriction endonuclease BsaI. We generated plasmids for C- as well as N-terminal tagging with GFP, mRFP and 3xFLAG. For gene deletion, we provide five different donor vectors for selection marker cassettes. These include standard cassettes for hygromycin B, nourseothricin and phleomycin resistance genes as well as FLP/FRT-based marker recycling cassettes for hygromycin B and nourseothricin resistance genes. To make cloning most feasible, we provide robust protocols, namely (1) an overview of cloning procedures described in this paper, (2) specific Golden Gate reaction protocols and (3) standard primers for cloning and sequencing of plasmids and generation of deletion cassettes by PCR and split-marker PCR. We show that our vector set is applicable for the biotechnologically relevant Penicillium chrysogenum and the developmental model system Sordaria macrospora. We thus expect these vectors to be beneficial for other fungi as well. Finally, the vectors can easily be adapted to organisms beyond the kingdom fungi.

质粒的克隆既费时又费钱。然而，克隆是许多基因功能分析标准实验的先决条件，包括缺失突变体的产生和基因产物的定位。在这里，我们提供了用于快速简便地克隆基因融合的 Golden Gate 载体以及适用于多种真菌的基因缺失载体。在 Golden Gate 克隆中，限制和连接在一锅反应中同时发生。我们的载体集包含常用类型 IIS 限制性内切酶Bsa 的识别位点I. 我们用 GFP、mRFP 和 3xFLAG 生成了用于 C 端和 N 端标记的质粒。对于基因缺失，我们为选择标记盒提供了五种不同的供体载体。这些包括潮霉素 B、诺丝丝菌素和腐草霉素抗性基因的标准盒，以及潮霉素 B 和诺丝丝菌素抗性基因的基于FLP/ FRT的标记回收盒。为了使克隆最可行，我们提供了可靠的协议，即 (1) 本文中描述的克隆程序概述，(2) 特定的金门反应协议和 (3) 用于质粒克隆和测序以及生成缺失盒的标准引物通过 PCR 和分裂标记 PCR。我们表明我们的向量集适用于生物技术相关的Penicillium chrysogenum和发育模型系统Sordaria macrospora。因此，我们希望这些载体对其他真菌也有益。最后，载体可以很容易地适应真菌界以外的生物。