Get Help Sign In

Cloning Strategies: Golden Gate cloning

Golden Gate cloning is known for being a scarless method that is suitable for assembling multiple DNA fragments for large constructs.

Numerous molecular cloning techniques are currently available that allow for the insertion of a specific DNA fragment, or in some cases more than one fragment, into a vector of choice.  Some of these cloning methods employ a restriction enzyme, which recognizes specific base pair sequence and cuts both DNA strands on both the vector and insert to create compatible ends, allowing the two to be joined together by DNA ligase. One of these methods is Golden Gate cloning.

What is Golden Gate cloning?

Golden Gate cloning utilizes Type IIS restriction enzymes to cut the insert and vector outside of their recognition site allowing for several fragments to then be assembled together with the vector in one reaction while at the same time also avoiding the formation of a scar in the final construct [1].  The overhangs produced using a Type IIS restriction enzyme are independent of the recognition site and unique to the sequence of the DNA fragments so after ligation no residual bases of the recognition sequence will be present in the cloned construct, preventing a so-called recognition site scar [1].

Because different Type IIS restriction enzymes cut the DNA at different locations outside of their recognition site, overhang sequences of differing lengths can be produced.  By carefully designing the sequences based on the IIS restriction enzymes to be used, multiple adjacent fragments can be assembled together into a vector in a single ligation reaction [2]. To accomplish this, it is important to design DNA fragments with Type IIS recognition sites at the terminal ends that overlap with the adjacent fragments.

Overview of the Golden Gate cloning method
Figure 1. An overview of the Golden Gate cloning method. The Golden Gate cloning method inserts more than one target DNA sequence into a vector. The vector and the DNA fragments, designated as fragments A and B, contain recognition sites (black) and cut sites (orange, navy blue, as well as purple to join the two fragments together). Both vector and DNA fragments are digested with Type IIS restriction enzymes, which generate a short nucleotide sequence overhang. The digested vector and the DNA fragments do not include the recognition sites, as shown in the figure. The overhang regions (designated as orange, navy blue, and purple in the figure) ligate to form an assembled construct.

Applications of Golden Gate cloning

Golden Gate cloning is scarless and can be used for cloning single inserts, assembling large constructs originating from multiple fragments, as well as assembling fragments that contain internal structural complexities [1].  It is considered an efficient method that can be done in a single tube and, as it works better than some other methods with a wide range of fragment lengths, it lends itself to different levels of assembly.  For example, it can be used to assemble several short genetic elements together to create a single gene, such as promoters, open reading frames, and terminators, or can be used to rapidly construct multiple genes into a vector simultaneously.  It can even be used in to generate multigene constructs from a library of assembled genes [3].


Areas that will not work well for Golden Gate cloning include sequences that contain GC extremities or secondary structures in the overlap regions. Although it can work better with repeat sequences than other scarless cloning methods that rely on homology alone, for Golden Gate assembly to work, it is critical that the Type IIS enzyme recognition site is uniquely present in the cloning site and does not appear anywhere else in the DNA sequence. If the recognition site occurs anywhere else in the insert or the vector, it will need to be removed to prevent undesired cuts. If there is an overhang with similar sequences (e.g., only 1 base pair difference) there is the chance that the vector may re-ligate which will decrease the cloning efficiency.

Design Considerations

  • When designing DNA fragment(s) for Golden Gate cloning, include the cut sequence of the Type IIS enzyme in the terminal regions of the insert. Ideally, sequences containing 4-base overhangs have the highest accuracy. Add the restriction enzyme recognition site terminal to the cut site. Make sure the recognition and cut sites are also added to the vector so that the overlap joins together.
  • Some enzymes require a spacer sequence between the recognition site and the cut site. For restriction enzymes that cut the sense strand one base pair outside of the recognition site, include a one base pair spacer nucleotide after the recognition sequence and before the cut sequence. For enzymes that cut after two base pairs, 2 base pair spacer nucleotides need to be included, and so on.
  • Also, a 4–5 base pair flanking sequence facilitates the recruitment of enzymes to the DNA template. Hence, add such a sequence to both terminal ends of the fragment to increase reaction efficiency.
  • Alternatively, plasmids can be used as starting material for Golden Gate cloning reactions. IDT can provide requested sequences in cloning vectors that are suitable for Golden Gate cloning (i.e. no Type IIS restriction sites present outside of the cut site).

Restriction enzyme s-recognition site vs cut site
Figure 2. The restriction enzyme’s recognition site differs from its cutting site. A Type IIS restriction enzyme cuts the non-specific nucleotide sequence away from its recognition site in the DNA molecule.


Additional Cloning Resources

  • Learn more about how DNA cloning and IDT gene fragment solutions can enable your research, helping you get your gene of interest on our DNA Cloning applications page.
  • Our DNA Cloning Guide provides techniques and tips for sequence design, codon optimization, and more for your cloning experiments.
  • Check out the Custom Gene Synthesis product page for an easy-to-use solution that allows you to skip in-house cloning steps and move quickly into functional studies with 100% sequence-verified clonal DNA.


  1. Bird JE, Marles-Wright J, Giachino A. A User's Guide to Golden Gate Cloning Methods and StandardsACS Synth Biol. 2022;11(11):3551-3563. 
  2. Engler C, Marillonnet S. Golden Gate cloningMethods Mol Biol. 2014;1116:119-131.
  3. Grützner R, Marillonnet S. Generation of MoClo Standard Parts Using Golden Gate CloningMethods Mol Biol. 2020;2205:107-123. 


Published Oct 3, 2023