Golden Gate Assembly Wizard

Ashu Singhal

Continuing toward our goal of making cloning painless, we're releasing a new method in our assembly wizard that many scientists have requested: Golden Gate Assembly.

We built this tool in conjunction with scientists from New England Biolabs, who offered their scientific expertise to help us formulate how the wizard should work. Stay tuned for further updates about how our partnership with NEB will create better tools for researchers.

Golden Gate uses Type IIs restriction enzymes which make 4 bp sticky ends next to their recognition sites. As long as the sticky ends can't improperly anneal, multiple fragments can combined in a single pot with high efficiency.

Selecting appropriate sticky ends by hand is a time-consuming and error-prone process. Our assembly wizard automates sticky end design and supports more complicated techniques such as including spacers between fragments and site-directed mutagenesis.

Below is a quick description of how to use the tool. As a reminder, our assembly wizard also supports cloning through Gibson and digest-and-ligate methods.

Creating a new assembly

To start using the assembly wizard, open any sequence in Benchling's editor and create a new assembly from the "Assembly Wizard" button at the bottom right.


You can set each fragment from a pair of type IIs cut sites or from any sequence selection. In the latter case, the wizard automatically generates primers that introduce recognition sites and valid sticky ends for the selected enzymes. Add spacer sequences using the icon next to each fragment.

When you're done selecting fragments, click the Assemble button to generate the output plasmid.

Viewing and adjusting the output plasmid

The Assembly tab in the output plasmid contains information about the primers that were designed. Buttons at the top help you export the primers and print a summary for your lab notebook.


To add point mutations, simply select any bases covered by a primer on the sequence map and type to replace. The relevant primers are automatically updated to incorporate the change, and mismatches with the parent sequence are labeled. Benchling versions the sequence and primers so you can undo any edit you make.


When you're done adjusting the design, select a folder where the primers should be saved and click "Finalize" from the Assembly tab. This will fully save your plasmid and enable all of Benchling's features on it, such as sequence alignment for verification.

As always, feedback on this new feature is welcome. If you have suggestions or would like to see a new assembly technique incorporated, drop us a line at Stay tuned for further updates, such as combinatorial assembly that allows you to set a library of parts for each fragment and generate all possible combinations in a single click.

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