Golden Gate Cloning in Geneious Prime

This Golden Gate cloning series shows how to simulate cloning in Geneious Prime using Type IIS enzymes such as BsaI, which cut outside their recognition site to create custom 4 bp overhangs for scarless, ordered assembly. Check parts for internal Type IIS sites, arrange fragments, and design primers, or batch clone multiple parts from a sequence list. Two videos, 2 to 6 minutes each.

Course Content

Perform Golden Gate Cloning

Batch Golden Gate Cloning

Perform Golden Gate Cloning

Check parts for internal Type IIS sites, then simulate Golden Gate cloning with a compatible enzyme and backbone vector. Reorder fragment tags, fix orientation or primer boundaries as needed, and export the final primers as CSV for ordering.

LENGTH 6 minutes

Batch Golden Gate Cloning

Batch clone multiple Golden Gate parts from a sequence list in one run, provided sequences share orientation and overhangs. Reorder parts in the construct layout panel and preview the number of possible product combinations before running.

LENGTH 2 minutes

Common Questions about Golden Gate Cloning

Which Type IIS restriction enzymes does Geneious Prime support for Golden Gate cloning?

Geneious Prime's Golden Gate tool uses a single Type IIS restriction enzyme per reaction ( such as BsaI ) consistent with standard Golden Gate protocols. The software includes pre-loaded commercially available enzyme sets for Golden Gate and BioBrick systems. Users select their enzyme before beginning; the tool then automatically detects existing cut sites, designs fusion boundaries, and generates PCR primers where needed.

Does Golden Gate cloning in Geneious Prime produce scarless assemblies?

Golden Gate cloning in Geneious Prime simulates the scarless, ordered assembly that the method produces in the lab. By cycling between Type IIS restriction and ligation in a single tube, Golden Gate leaves no restriction site scar at junction points. Geneious Prime models this outcome and displays the final concatenated construct with annotations confirming that all junctions are correctly formed.

How does Geneious Prime determine overhang boundaries in Golden Gate assembly?

Geneious Prime automatically detects existing Type IIS restriction sites, overhang annotations, primer_bind annotations, and blunt ends in input sequences. It then applies six precedence rules to determine fusion site boundaries. Researchers can override default boundaries through the interface dropdown, giving full control over overhang placement while benefiting from automated detection of existing site architecture.

Can I export Golden Gate primers from Geneious Prime for synthesis ordering?

Geneious Prime lets you save designed Golden Gate primers and export them to CSV format for direct submission to an oligonucleotide synthesis provider. The final construct displays concatenated sequence annotations confirming successful assembly, and intermediate PCR products can also be exported for custom synthesis where needed.

Does Geneious Prime support batch Golden Gate cloning?

Geneious Prime supports batch Golden Gate cloning using sequence lists, allowing multiple parts to be assembled simultaneously. The tool automatically arranges parts based on compatible overhangs and tags each part to indicate whether it uses an existing cut site or requires a PCR product streamlining the planning of multi-fragment assemblies.

How is Golden Gate cloning in Geneious Prime different from restriction cloning?

Golden Gate cloning uses Type IIS restriction enzymes that cut outside their recognition sequence, producing custom 4-bp overhangs that enable ordered, scarless assembly of multiple fragments in a single reaction. Restriction cloning in Geneious Prime uses conventional Type II enzymes that cut within their recognition site, leaving defined restriction site sequences at junctions. Both workflows are supported in Geneious Prime through a shared cloning interface, allowing researchers to choose the approach that fits their construct design.