The article introduces a new cloning method called 'Golden Gate' cloning, which allows for the rapid and precise transfer of DNA fragments from an entry clone into an expression vector in a single step and a single tube. This method uses type IIs restriction enzymes, which cut outside their recognition sites, enabling the creation of unique DNA overhangs that can be ligated together without leaving any unwanted sequences in the final construct. Unlike traditional site-specific recombination-based cloning methods, which leave recombination site sequences in the final product, 'Golden Gate' cloning produces recombinant plasmids without these sequences, providing greater precision in genetic manipulation. The method is highly efficient, with nearly 100% correct recombinant plasmids obtained after just 5 minutes of restriction-ligation. This efficiency is achieved by designing the cleavage sites of the restriction enzymes so that the resulting fragments can be ligated together without the original restriction sites. The method was tested with various DNA fragments and was found to be effective in cloning multiple inserts into a single expression vector. The strategy also allows for the elimination of internal restriction sites in the gene of interest, ensuring that no unwanted sequences are present in the final product. The 'Golden Gate' cloning method is particularly useful for high-throughput cloning applications, such as the expression of large sets of open reading frames (ORFs) in plants. It is compatible with various expression vectors and can be used to transfer genes of interest into different expression systems. The method is also advantageous because it does not require the use of commercial kits or components, making it more cost-effective for large-scale projects. Overall, the 'Golden Gate' cloning method offers a simple, efficient, and precise alternative to traditional cloning techniques, making it a valuable tool in genetic engineering.The article introduces a new cloning method called 'Golden Gate' cloning, which allows for the rapid and precise transfer of DNA fragments from an entry clone into an expression vector in a single step and a single tube. This method uses type IIs restriction enzymes, which cut outside their recognition sites, enabling the creation of unique DNA overhangs that can be ligated together without leaving any unwanted sequences in the final construct. Unlike traditional site-specific recombination-based cloning methods, which leave recombination site sequences in the final product, 'Golden Gate' cloning produces recombinant plasmids without these sequences, providing greater precision in genetic manipulation. The method is highly efficient, with nearly 100% correct recombinant plasmids obtained after just 5 minutes of restriction-ligation. This efficiency is achieved by designing the cleavage sites of the restriction enzymes so that the resulting fragments can be ligated together without the original restriction sites. The method was tested with various DNA fragments and was found to be effective in cloning multiple inserts into a single expression vector. The strategy also allows for the elimination of internal restriction sites in the gene of interest, ensuring that no unwanted sequences are present in the final product. The 'Golden Gate' cloning method is particularly useful for high-throughput cloning applications, such as the expression of large sets of open reading frames (ORFs) in plants. It is compatible with various expression vectors and can be used to transfer genes of interest into different expression systems. The method is also advantageous because it does not require the use of commercial kits or components, making it more cost-effective for large-scale projects. Overall, the 'Golden Gate' cloning method offers a simple, efficient, and precise alternative to traditional cloning techniques, making it a valuable tool in genetic engineering.