Gibson Construct Records: Overlaps Scars and Fragment Ids

A documentation checklist for Gibson assembly constructs is a record gate before the bench run, not a wet-lab implementation walk. The outcome is a construct record that names every fragment, every overlap, and every scar the assembly will create. Inputs are the source sequence versions and the primers that add the overlaps. A gel photograph in a shared folder is not that record. The Gibson implementation checklist still owns the pot, ratios, and colony screen. Gibson simulation review still owns the in-silico preview. Assembly-method choice still owns chemistry. This page only writes the fields the later steps must cite.

Record Overlaps, Scars, and Fragment IDs Before the Bench Run

The search is how to document Gibson assembly constructs. NEB’s Gibson Assembly manual describes the anatomy this record must capture: a 5′ overlap homologous to the neighboring fragment and a 3′ priming region that has to PCR. Suggested overlap length in that manual is a vendor heuristic (15–25 nt with a Tm at or above 48 °C under their simple rule), not a Zetta specification and not a promised efficiency. Write the actual overlap sequence you will use. After those fields exist, ZettaGene can show a Gibson simulation on the named map. Official product pages list restriction, Gibson, and homologous-alignment simulation. Golden Gate is not a Zetta feature and is not a substitute record for Gibson overlaps.

Steps: Write the Gibson Construct Record

1. Assign a construct ID and the expected topology (circular plasmid or linear) before any fragment is ordered. Checkpoint: the ID is not “Gibson this week.”
2. List each fragment as a source ID plus version: verified plasmid, PCR template, or synthetic piece. A generic part name is not an ID. Checkpoint: every fragment resolves to one sequence version.
3. Write each overlap as sequence, length, and which two fragment ends it joins. If a primer adds the overlap, record the full oligo, not only the gene-specific core. Checkpoint: a reviewer can rebuild the junction from the record alone.
4. Name the scar or seamless junction each overlap will leave. “No scar” is allowed only when the overlap is homologous sequence that remains in frame as intended. Checkpoint: translation across the junction is written if a CDS crosses it.
5. State orientation and order. Multi-fragment Gibson fails when two overlaps are interchangeable. Checkpoint: the neighbor of each end is unique.
6. Attach the expected full-length sequence and the features that must survive: marker, origin, and any CDS that crosses a seam. Checkpoint: the record points to one expected map, not a folder of drafts.
7. Write the verification plan this record will later accept: junction Sanger, whole-plasmid sequence, or a diagnostic digest that can actually see the new seams. Checkpoint: the plan names the junctions, not “sequence it.”

Expected Result

A second person can list fragment IDs, overlap sequences, scars, order, and the expected map without asking the designer. Opening the simulation tool is not verification. Pouring the assembly is not verification. The record has to exist first.

Verification

Read the record as a materials list. If any fragment lacks a version, any overlap lacks a sequence, or any CDS-crossing seam lacks a frame sentence, documentation is incomplete. If the next job is the wet protocol, open the implementation checklist. If the next job is still the virtual preview, open the simulation review. Do not use this page to invent Golden Gate fields as Zetta capabilities.

Common Failure Modes

The usual fail is a primer name without the 5′ tail. The second fail is two fragments that share a similar overlap so the pot can assemble in more than one order. The third fail is a gel of “the right size” with no record of which scar was expected. A wet-lab checklist that names ratios and incubation times still needs this record to know which overlaps were intended. Prefer a short junction table over a paragraph that says the assembly “looked fine.” ZettaGene is the Gibson map after the fields are named. It does not replace the bench checklist and it does not make Golden Gate a Zetta product.

Frequently Asked Questions

Is a Gibson gel photo enough documentation?

No. A band does not name fragment versions, overlap sequences, or scars. Record those fields before the pot is mixed.

Does a Gibson simulation replace the construct record?

No. Simulation previews the map. The record is the citable list of IDs, overlaps, and expected junctions the bench and the reviewer will use.