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tooluniverse-molecular-cloning

Designs Gibson and Golden Gate assembly junctions for joining DNA fragments into constructs.

1.7k|254|Updated Mar 3, 2025
Search Tags:#synthetic biology#dna assembly#golden gate#molecular cloning#cloning qc#gibson assembly#overhang design
One-click install
npx skills add https://github.com/mims-harvard/ToolUniverse --skill tooluniverse-molecular-cloning
Or copy as Structured Prompt for Agent▼
Please help me install this Agent Skill.
Skill: tooluniverse-molecular-cloning
Source: https://github.com/mims-harvard/ToolUniverse/tree/main/plugins/tooluniverse/skills/tooluniverse-molecular-cloning
Command: npx skills add https://github.com/mims-harvard/ToolUniverse --skill tooluniverse-molecular-cloning

SYSTEM DOCUMENTATION & REQUIREMENTS

💡 This Skill includes scripts (resource) components.

What problem does it solve?

Planning how to join DNA fragments into a construct requires careful design of assembly junctions, and failures from internal restriction sites, non-unique overhangs, or unbalanced overlap Tm values waste time and reagents. This Skill designs Gibson Assembly overlaps and Golden Gate Type IIS overhangs and screens parts for the common failure modes before you order oligos.

Core Features & Use Cases

  • Gibson Assembly Design: Generates 15-40 bp homology overlaps with balanced Tm for seamless multi-fragment joining, returning fragments extended with the arms to add to PCR primers.
  • Golden Gate Design: Assigns unique, non-palindromic 4-bp fusion overhangs for BsaI/BbsI Type IIS assembly and flags internal recognition sites that require domestication by silent mutation.
  • Assembly QC Script: The included cloning_qc.py script screens parts for internal Type IIS sites, overhang uniqueness and palindromes, and Gibson overlap length/GC content, reporting PASS/WARN flags.
  • Use Case: A researcher assembling a three-part expression cassette runs the Golden Gate design to get unique overhangs, then QC-checks every part for internal BsaI sites before ordering primers.

Quick Start

Ask the AI to design a Gibson assembly for your DNA fragment sequences and QC the resulting overlaps for length and GC content.

Frequently Asked Questions about tooluniverse-molecular-cloning

High-intent search queries and answers about installing and using this skill.

FAQPage Schema
How do I design a Gibson Assembly for multiple DNA fragments?▼

Provide your fragment sequences in assembly order to the Gibson design operation with an overlap length of 20-25 bp. It returns left and right homology overlaps plus fragments extended with the arms you add to your PCR primers.

Gibson Assembly vs Golden Gate: which cloning method should I use?▼

Use Gibson for a few fragments needing seamless junctions at arbitrary positions. Use Golden Gate for many standardized parts, combinatorial libraries, or repeated one-pot assemblies where you can remove internal BsaI/BbsI sites.

Why does Golden Gate assembly fail with internal BsaI sites?▼

The Type IIS enzyme cuts at every recognition site, including ones inside your parts, fragmenting them during assembly. You must domesticate each part by silently mutating internal BsaI (GGTCTC) or BbsI (GAAGAC) sites before assembly.

What makes a good 4-bp overhang for Golden Gate cloning?▼

Each overhang must be unique, non-palindromic (not equal to its own reverse complement), and not the reverse complement of another overhang in the set. Avoid high-GC or all-AT overhangs; high-fidelity sets like Potapov 2018 ligate most cleanly.

What are the limitations of in silico cloning assembly design?▼

These tools design junctions but do not simulate the ligation or exonuclease reaction or guarantee assembly efficiency. They also do not check vector backbone compatibility or ORF reading frames, so validate constructs by sequencing.

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