What overhang length does Golden Gate use?

Four nucleotides by convention for BsaI and BsmBI. Some enzymes such as SapI or AarI leave three, and BbsI leaves four with a different spacing.

Why must the next part's overhang be the reverse complement?

Because the two overhangs anneal as antiparallel strands. If part A ends with AATG on the top strand, part B must begin with CATT on its top strand for the two to pair.

What is a scar in Golden Gate?

The 4 bp block left at a junction. Depending on how the overhangs were chosen the scar may be a neutral spacer or may introduce an unwanted codon — worth checking when fusing coding sequences.

Can I reuse the same overhang twice in one reaction?

No. Repeated overhangs let parts assemble in the wrong order or drop out. Every junction in a single reaction needs a distinct overhang set.

Golden Gate Assembly Tool

Assemble Type IIs parts in a defined order — check every 4 nt overhang and see the scar each junction leaves.

Fragment 1

Left overhang (5'→3')

Right overhang (5'→3')

0 bp

Fragment 2

Left overhang (5'→3')

Right overhang (5'→3')

0 bp

Paste at least two fragments, in the order you want them joined.

Description

Give each part a 5′ and 3′ overhang (5′→3′). The tool checks that adjacent overhangs can ligate, reports the scar a junction recreates, and returns the assembled sequence with the overhangs merged.

How to use

Enter parts in the order you want them in the final construct. The left overhang of a part must be the reverse complement of the right overhang of the part before it — the junction table tells you where that fails.

Learn more

What it does

Golden Gate assembly uses a Type IIs enzyme such as BsaI or BsmBI that cuts outside its recognition site, releasing parts with user-defined 4 nt overhangs. Because the recognition site is removed by cutting, digestion and ligation can run in one tube to completion. This tool models the sequence outcome of that reaction.

How it works

Part i is joined to part i+1 when the right overhang of the first and the left overhang of the second are complementary — in practice the second is the reverse complement of the first. When the two overhangs are identical strings, ligation recreates the full 4 nt block and the junction leaves that sequence as a scar; the tool names it so you can see exactly what your fusion adds.

Worked example

A promoter with right overhang AATG followed by a CDS whose left overhang is CATT (the reverse complement) gives a compatible junction. Because AATG and CATT are not the same string, no bases are duplicated at the joint — the assembled sequence simply continues.

When to use it

Use it when building MoClo / GoldenBraid style transcriptional units, when reusing a library of standardised parts, or when debugging an assembly that gave the wrong colony count. Standard design practice is to avoid overhangs that are palindromic or within one base of another in the same reaction.

FAQ

What overhang length does Golden Gate use?: Four nucleotides by convention for BsaI and BsmBI. Some enzymes such as SapI or AarI leave three, and BbsI leaves four with a different spacing.
Why must the next part's overhang be the reverse complement?: Because the two overhangs anneal as antiparallel strands. If part A ends with AATG on the top strand, part B must begin with CATT on its top strand for the two to pair.
What is a scar in Golden Gate?: The 4 bp block left at a junction. Depending on how the overhangs were chosen the scar may be a neutral spacer or may introduce an unwanted codon — worth checking when fusing coding sequences.
Can I reuse the same overhang twice in one reaction?: No. Repeated overhangs let parts assemble in the wrong order or drop out. Every junction in a single reaction needs a distinct overhang set.

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