Gibson cloning

Use this page when you want to assemble a backbone and one or more inserts by overlap, check the junction quality, and create the final construct in one workflow.

Before you start

  • Open a construct with valid sequence content.
  • Make sure any reusable inserts or cut fragments are available in your workspace Projects or Parts.

Switch to cloning view and stage the assembly

  1. Open the construct workspace and switch the main view to Cloning.
  2. In the Technique picker, choose Gibson.
  3. Set the backbone source to the current plasmid or to a reusable cut plasmid from the library.
  4. Add one or more inserts with the staging controls and confirm their order and orientation.

Gibson staging with backbone and inserts visible

Set overlap constraints and run the simulation

In Gibson settings, review the controls that affect the QC state:

  • Auto-size overlaps
  • Min overlap length
  • Min overlap Tm
  • optional Max overlap length

Then click Simulate and wait for the preview to populate.

Review overlap QC and junction details before creation

  1. Open the Junction QC & primers area and inspect the selected junction.
  2. Review the Overlap QC summary for pass or fail status.
  3. Check whether the primer actions or compatibility notes change based on the staged junction.
  4. If the overlap is weak, adjust length, Tm target, insert order, or orientation and run Simulate again.

Overlap QC and junction review for Gibson assembly

Create the assembled plasmid

  1. Confirm the preview map reflects the construct you intended to build.
  2. Click Create plasmid....
  3. Enter the plasmid name and confirm creation.
  4. Open the new record in Sequence or Circular Map view for a final review.

Create plasmid dialog after Gibson simulation

If it doesn't look right

  • Simulate is disabled: Confirm that a backbone and at least one insert with real sequence are staged.
  • Overlap QC fails: Increase the overlap length, revisit the Tm target, or change the insert order before creating the plasmid.
  • The create action has no usable preview: Run Simulate again and check the junction review first.