Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.

中文翻译：

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通过新的整合位点扩展了酿酒酵母基因组的EasyClone-MarkerFree工具包。

生物技术生产需要遗传稳定的重组菌株。为了确保基因组稳定性，通常将重组DNA整合到宿主菌株的基因组中。已经开发出多种遗传工具以将基因组整合到面包酵母中。以前，我们已经开发了矢量工具包EasyClone-MarkerFree，用于稳定整合到啤酒酵母X，XI和XII染色体上的11个位点。CRISPR-Cas9系统实现了无标记整合。在这项研究中，我们用位于不同染色体上的八个其他基因间整合位点扩展了该试剂盒。新站点的集成效率超过80％。所有八个位点的绿色荧光蛋白（gfp）的表达水平与原始EasyClone-MarkerFree工具包中的XI-2位点相似或更高。细胞生长不受新八个位置中任何一个位置整合的影响。八载体扩增试剂盒可从AddGene获得。