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For detailed Golden Gate protocols, complete with helpful tips and tricks, see The Sainsbury Lab website or Engler & Marillonet. This allows for multiple DNA components (promoters, genes, terminators, etc) to be assembled in the correct order in a single reaction. Scientists can engineer unique enzyme recognition sites that flank their DNA fragment in an inverse orientation. As with all Golden Gate-based methods, this system exploits the ability of Type IIS enzymes to cut outside their recognition site and permits DNA fragments with compatible overhangs to be efficiently assembled. Sometimes referred to as MoClo, this strategy uses the Type IIS restriction enzymes BsaI and BpiI/BbsI to efficiently assemble up to six DNA fragments at a time. Synthetic biologists have leveraged the power of Golden Gate cloning into a modular cloning strategy. Golden Gate assembly is also less expensive than many commercial cloning methods. The popular Gateway cloning system produces constructs with an attB recombination scar encoding eight amino acids, but Golden Gate assembly can be designed to be scarless. Exonuclease-based methods like Gibson assembly require 20-40 bp of homology at the ends of DNA fragments to specify assembly order, so fragments with 5’ or 3’ sequence homology cannot be assembled using this method, but can be assembled with Golden Gate. Golden Gate assembly has a few advantages over other cloning methods. Although efficiency may decrease with an increased number of fragments, or the ligation of very small/very large fragments, these problems can be overcome by screening a higher number of potential clones. Unique 4 base overhangs can be used to assemble multiple fragments - up to 10 fragments are commonly assembled in a single reaction! These overhangs specify the desired order of fragments, and the loss of enzyme recognition sites after ligation favors formation of the construct of interest. Another strength of Golden Gate cloning is its scalability. As a result, the ligation process is close to 100% efficient. In contrast, formation of the desired ligation product is irreversible because this construct does not retain the enzyme recognition sites. Although the original destination vector + insert may spontaneously religate, this transient construct retains functional Type IIS sites and will be re-digested. The destination vector and entry vector(s) are placed in a single tube containing the Type IIS enzyme and ligase. Golden Gate cloning is one of the easiest cloning methods in terms of hands-on time, as digestion and ligation can be done in one 30-minute reaction. Entry DNA overhangs may be present in the original plasmid (Option 1) or added using PCR-based amplification (Option 2). As shown below, a fragment with 5’ overhang TGGA and 3’ overhang TCCG can be ligated into a vector containing those overhangs. The destination vector contains sites with complementary overhangs that direct assembly of the final ligation product. As a result, these sites are eliminated by digestion/ligation and do not appear in the final construct. The cloning scheme is as follows: the gene of interest is designed with Type IIS sites (such as BsaI or BbsI), that are located on the outside of the cleavage site. When designed correctly, the recognition sites do not appear in the final construct, allowing for precise, scarless cloning. Since these overhangs are not part of the recognition sequence, they can be customized to direct assembly of DNA fragments. Type IIS restriction enzymes are unique from "traditional" restriction enzymes in that they cleave outside of their recognition sequence, creating four base flanking overhangs. Golden Gate cloning technology relies on Type IIS restriction enzymes, first discovered in 1996. We’ll walk you through how to apply this precise and easy-to-use system to your cloning efforts. Each method has its own pluses and minuses, but Golden Gate cloning has been especially useful within both the synthetic biology and genome engineering fields. Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods.