Sequence-level Design Review: A Checklist Before You Build

An implementation checklist for sequence-level construct review is a base-readout gate, not a meeting agenda and not a purchase-order packet. Freeze the design only after someone has walked fusion frames, named every scar or its absence, and searched sites that would break the later cut or assembly. A colored map can lie. Construct planning owns the pre-order freeze of features and reviewers. Gene-synthesis order review owns the later PO. CRISPR pre-cloning review owns PAM, marker, and promoter.

What This Review Must Freeze

The search is sequence-level design review checklist. The object is the sequence, not the cartoon. A plasmid-map reading guide tells reviewers to check insert boundaries and reading frame at fusion junctions against the bases. An in-frame arrow on the drawing is not that check. Work on scarless assembly treats leftover fusion sites at CDS boundaries as functional constraints on mRNA and ribosome binding, not as decoration. Use that warning. Do not import Golden Gate as a Zetta product. Official simulation on Zettalab pages is restriction, Gibson, and homologous alignment.

This gate freezes three things: frame at every fusion, every scar or a written statement that the junction is scarless, and sites that would make the next wet step illegal. It does not freeze reviewer roles. That is the team map-review checklist.

Sequence-Level Review Steps

1. Open the construct as sequence, not only as a circular cartoon. Record the file name, length, and topology.
2. Walk each fusion or tag junction in the reading frame that the biology claims. Write the codon coordinates. Reject the review if the frame is inferred from the drawing alone.
3. Mark every assembly leftover or write “no scar” at each junction. Treat a CDS-boundary scar as a design decision, not as noise.
4. Search forbidden sites for the intended next step: an internal cutter that would break a cloning digest, a Type IIS site left from a method you are not running in ZettaGene, or a primer-binding collision.
5. Write freeze or reject with a named reviewer and a date. A silent “looks good” is not a record.

ZettaGene is the sequence view used in this review after the gate is understood. It does not replace the walk. Do not ask the workspace to simulate Golden Gate. That method is not listed on the official product page. A forbidden site is a site that makes the next intended wet step illegal, not every cutter on a crowded map. If the next step is a diagnostic digest, the illegal site is one that would hide the expected band. If the next step is a cloning cut, the illegal site is an extra hit inside the insert or a missing unique site in the backbone.

Expected Result and Verification

The expected result is a short note that a second person can audit: junction coordinates with frame stated, scars named or marked absent, and forbidden-site search dated. Verification fails if the only artifact is a screenshot of a colored map. Verification also fails if oligos or a synthesis PO already left the lab. In that case you are reconstructing, not reviewing. Send the purchase packet to the synthesis-order sibling if that is the job that remains.

Common Failures Before Anyone Orders

Unnamed junctions are the usual miss. Assumed in-frame tags are next: the map draws a fusion the sequence does not contain. Ignored leftovers from a modular method sit in the CDS and change translation without changing the cartoon much. A planning freeze that never opened the sequence is not this checklist — it belongs on the planning page. A CRISPR map that still has not shown PAM, marker, and promoter is not this checklist either. Close those objects on the CRISPR pre-cloning page, then return here if the same file still needs a frame walk.

Frequently Asked Questions

Is a colored plasmid map enough to close a sequence-level design review?

No. The diagram can show an in-frame fusion the sequence does not have.

Is this the same checklist as a gene-synthesis order review?

No. Synthesis-order review owns the purchase packet. This page owns the base readout before that packet exists.