How to use the Interactive Construct Builder

Use the builder to inspect and adjust antigen boundaries from each protein report. Reports with structure assets connect sequence, AlphaFold confidence, PAE, ortholog mappings, cysteine warnings, furin-site warnings, and export-ready sequence records.

What the builder shows

Each protein report page includes the target sequence and a Live Selected Region table. Reports with a local AlphaFold model also include the structure, an interactive pLDDT trace, and an interactive PAE matrix.

Selections stay synchronized across the panels present in the report. Selecting residues in the sequence or plots highlights the same residue window in the structure and updates the TSV/FASTA outputs.

Basic workflow

Start from the construct classes present in the report: full design region, PDB-backed constructs, annotated domains, strict calculated regions, then lenient calculated regions.
Select or adjust a residue window in the sequence, pLDDT plot, or PAE plot.
Evaluate compactness, model confidence, and biological context for the selected region.
Review warnings for unpaired cysteines and furin-like cleavage sites.
Copy the TSV or FASTA output for cloning, ordering, or downstream analysis.

Live Selected Region

The table reports export-ready sequence records for the current selection: human construct name, boundaries, sequence, and equivalent mouse and cynomolgus monkey regions where ortholog mappings exist.

The construct name follows the format GENE_SPECIES_start-end. Selecting optional Cys-to-Ser mutations adds mutation suffixes to the name and updates the sequence immediately.

Structure panel

Reports with a local AlphaFold model show the structure as a cartoon colored by AlphaFold confidence. Selected residues are highlighted for boundary review against folded domains, linkers, cysteines, and visible surface regions.

Cysteines are emphasized in yellow with side chains where present. Exposed or unpaired cysteines can complicate recombinant soluble antigen expression and purification.

How to interpret pLDDT

pLDDT is AlphaFold's per-residue local confidence estimate. It describes confidence in the immediate geometry around a residue. Domain-domain orientation requires PAE review.

pLDDT range	Practical interpretation	Construct-design value
90-100	Very high local confidence.	Strong support for including this residue in a structured construct, assuming topology and biology also make sense.
70-90	Generally confident local structure.	Often acceptable for domain cores and boundary-adjacent residues.
50-70	Low confidence or flexible geometry.	Review manually. This range often marks flexible linkers, uncertain loops, or boundary regions.
Below 50	Very low confidence, often disordered.	Consider trimming unless the region has a required biological role.

For soluble antigen design, high mean pLDDT and a high fraction of residues above the structured threshold support local fold confidence. Use PAE to evaluate relative orientation between domains.

How to interpret PAE

PAE, predicted aligned error, estimates how uncertain AlphaFold is about the position of one residue or region when another residue or region is aligned. Lower PAE means AlphaFold expects the relative placement of those residues to be reliable; higher PAE means the relative placement is uncertain.

PAE pattern	What it suggests	Construct-design value
Low PAE block within a region	The selected residues likely form a coherent structural unit.	Good support for a single-domain or stable multi-domain construct.
High PAE between two neighboring blocks	The blocks have uncertain relative placement or a flexible linker between them.	Consider splitting constructs at or near the linker.
Low pLDDT and high boundary PAE	A flexible boundary is likely.	Often useful for trimming or separating domains.
High pLDDT but high inter-domain PAE	Each domain has local fold support, with uncertain domain orientation.	Potentially expressible, with uncertain rigid antigen-surface presentation.

PAE matrices are asymmetric model-derived estimates. Use them to evaluate whether a selected region behaves like one cohesive unit or contains an internal flexible boundary.

Choosing boundaries

Place boundaries outside transmembrane helices, cytoplasmic tails, signal peptides, low-confidence disordered tails, processing sites, and obvious flexible linkers, except when the construct intentionally includes those regions.

Prefer boundaries that align with curated domains, PDB construct precedent, low local disorder, PTM-aware sequence context, and a clean PAE separation between domains.

Strict versus lenient constructs

Strict calculated constructs favor compact single-domain units with stronger confidence and cleaner boundaries. Lenient constructs retain larger coherent regions for multi-domain surfaces and larger conformational epitopes.

Warnings

Unpaired cysteine warnings flag cysteines associated with unwanted disulfides, aggregation, or expression heterogeneity. PTM warnings flag curated processing or cleavage annotations. Furin-site warnings flag basic motifs with cleavage risk in some producer cells or biological contexts.

Manual overrides

Use manual boundary choices for ligand-binding sites, obligate partners, required disulfides, epitope constraints, glycosylation, and literature-supported constructs.

Practical checklist

Is the region extracellular or intentionally membrane-associated?
Does the region have acceptable pLDDT across most residues?
Does the PAE matrix support the region as one coherent unit?
Do boundaries avoid low-confidence tails and flexible linkers?
Are unpaired cysteines, furin motifs, and key ligand-binding regions accounted for?
Do boundaries preserve curated PTMs or processing sites that matter for the target?
Do mouse and cynomolgus monkey equivalent sequences preserve the selected region if cross-species antibody screening is planned?

For the full methodology behind the pre-generated construct list, see the Pre-generated Constructs page.