A one-pot, one-step, precision cloning method with high throughput capability is described. This method allows for the efficient and accurate transfer of DNA fragments from an entry clone into an expression vector without leaving recombination site sequences in the final construct. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence, resulting in 5' or 3' DNA overhangs that can consist of any nucleotide. This property enables the development of a cloning strategy called 'Golden Gate' cloning, which allows for the rapid and efficient assembly of DNA fragments in a single ligation reaction. This method is as efficient as current recombination-based cloning technologies but produces recombinant plasmids that do not contain unwanted sequences, providing precision for genetic manipulation. The method involves designing the gene of interest to be flanked by BsaI sites, allowing for the elimination of the restriction site after subcloning. The recipient expression vector contains two BsaI sites that flank a LacZ alpha fragment and are positioned such that the recognition sites are eliminated after digestion. The method was tested with various constructs, including a GFP gene and a chloroplast gene, and showed high efficiency in cloning. The method allows for the subcloning of multiple DNA fragments from separate entry clones into an acceptor vector, with the only requirement being the design of compatible restriction sites in the entry clones and the acceptor vector. The method was also tested for the elimination of internal BsaI sites in the gene of interest, which can prevent the use of this strategy. This was achieved by designing primers that introduce mutations to eliminate the BsaI recognition sequence. The method was found to be highly efficient, with the ability to clone at least three DNA fragments from three separate entry clones into an acceptor vector. The method is suitable for use with a variety of expression vectors and can be used to transfer one gene of interest or any DNA fragment of interest from one entry clone to a series of different expression vectors designed to have compatible cloning sites. The method is efficient, simple to use, and provides high precision in genetic manipulation.A one-pot, one-step, precision cloning method with high throughput capability is described. This method allows for the efficient and accurate transfer of DNA fragments from an entry clone into an expression vector without leaving recombination site sequences in the final construct. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence, resulting in 5' or 3' DNA overhangs that can consist of any nucleotide. This property enables the development of a cloning strategy called 'Golden Gate' cloning, which allows for the rapid and efficient assembly of DNA fragments in a single ligation reaction. This method is as efficient as current recombination-based cloning technologies but produces recombinant plasmids that do not contain unwanted sequences, providing precision for genetic manipulation. The method involves designing the gene of interest to be flanked by BsaI sites, allowing for the elimination of the restriction site after subcloning. The recipient expression vector contains two BsaI sites that flank a LacZ alpha fragment and are positioned such that the recognition sites are eliminated after digestion. The method was tested with various constructs, including a GFP gene and a chloroplast gene, and showed high efficiency in cloning. The method allows for the subcloning of multiple DNA fragments from separate entry clones into an acceptor vector, with the only requirement being the design of compatible restriction sites in the entry clones and the acceptor vector. The method was also tested for the elimination of internal BsaI sites in the gene of interest, which can prevent the use of this strategy. This was achieved by designing primers that introduce mutations to eliminate the BsaI recognition sequence. The method was found to be highly efficient, with the ability to clone at least three DNA fragments from three separate entry clones into an acceptor vector. The method is suitable for use with a variety of expression vectors and can be used to transfer one gene of interest or any DNA fragment of interest from one entry clone to a series of different expression vectors designed to have compatible cloning sites. The method is efficient, simple to use, and provides high precision in genetic manipulation.