Golden Gate Assembly designer
Maintained by the Talindrew team · Last updated
A Golden Gate Assembly designer lays out a one-pot Type IIS assembly: which enzyme cuts each part, the 4 nt fusion sites that fix the order, and whether any site will fail. Talindrew's Golden Gate wizard builds Type IIS assemblies with fusion-site optimisation to minimise scars and runs a pre-flight check for overhang compatibility, single-cut verification and methylation blocking before you commit. A worked example loads with one click. For modular cloning, the cloning workspace loads kit templates such as MoClo YTK, assembles every valid combination from a syntax, and validates a custom overhang graph in its syntax builder.
Specifications
| Chemistry | Type IIS restriction and ligation, 4 nt fusion sites, scarless assembly |
|---|---|
| Fusion sites | Optimised to minimise scar sequences |
| Checks | Overhang compatibility, single-cut verification, methylation blocking |
| Try first | Worked example loads with one click |
| Kits | Kit templates from the OpenCloning submission repository, MoClo YTK included |
| Combinatorial | Assembler chains the categories and assembles every valid combination; results download as a zip |
| Custom syntaxes | Syntax builder with overhang graph validation, saved as JSON |
| Price | Free with a Google sign-in; runs in the browser |
How do I design a Golden Gate Assembly?
The order of the parts is set by their overhangs, so the design work is choosing fusion sites that are unique, compatible end to end, and not blocked by an internal recognition site or methylation.
- Add the parts and the destination vector in assembly order.
- Let the wizard assign the Type IIS enzyme and optimise the 4 nt fusion sites.
- Read the pre-flight check: incompatible overhangs, parts cut more than once, and sites blocked by methylation are flagged.
- Simulate the product, then save the design or move a modular build into the workspace kits and assembler.
Designer or protocol: which page do I need?
This page is the design tool. The Golden Gate Assembly guide covers the bench protocol (vector and insert amounts, buffer, enzyme mix and cycling programmes from NEB's BsaI-HFv2 kit) and the overhang design rules.
How should fusion sites be chosen?
Overhang choice decides whether a Golden Gate reaction assembles in the right order. Ligation fidelity data for all 256 possible 4 nt overhangs (Potapov et al., 2018) shows some pairs mis-ligate, so designs follow a few rules.
- Use overhangs that differ from each other by at least two nucleotides.
- Avoid palindromic overhangs, which ligate to themselves.
- Avoid overhang sets that are all AT or all GC.
- Do not reuse an overhang in one reaction unless those junctions are meant to be interchangeable.
- Remove internal BsaI or BsmBI sites from every part before assembly.
Frequently asked questions
Does it catch sites blocked by methylation?
Yes. The pre-flight check flags recognition sites blocked by methylation, alongside incompatible overhangs and parts that would be cut more than once, before you commit to the design.
Is the Golden Gate Assembly designer free?
Yes. The Golden Gate wizard, the kit templates and the assembler are part of Talindrew's free cloning suite in the browser, with a Google sign-in.
Can it build MoClo assemblies?
Yes. The cloning workspace loads kit templates from the OpenCloning submission repository, MoClo YTK included, and its assembler chains the part categories and assembles every valid combination at once.
Related
Try it on your own sequence.
Free to use. No download required. Works in any modern browser.