STRC AAV Vector Design
Recommended Vector Layout
5'ITR — B8 enhancer — IgK SP — mini-STRC CDS — bGH polyA — 3'ITR
145 587 63 3,228 225 145
Between ITRs: 4,103 bp / 4,700 bp limit = 597 bp headroom
With WPRE3 (247 bp short version): 4,350 bp = 350 bp headroom. Still fits.
Components
B8 Enhancer (587 bp)
- Prestin (SLC26A5) enhancer, OHC-specific expression
- From Zheng et al. (2020), proven in vivo for outer hair cell targeting
- Chosen over Myo7a (too large) and CMV (not cell-specific)
IgK Signal Peptide (63 bp / 21 aa)
METDTLLLWVLLLWVPGSTGD- Replaces native STRC SP (removed with N-terminal truncation)
- Validated by SignalP 6.0: 99.97% confidence (see STRC Signal Peptide Validation)
Shorter Mini-STRC CDS (3,228 bp / 1,076 aa)
- Residues 700-1775 of full STRC
- pTM 0.86, 4% disordered (AF3 job F)
- With stop codon: 3,231 bp
bGH PolyA (225 bp)
- Bovine growth hormone polyadenylation signal
- Standard in AAV vectors, compact and efficient
ITRs (145 bp each)
- AAV2 inverted terminal repeats
- Required for packaging and second-strand synthesis
Codon Optimization (2026-03-19)
Full protein: IgK SP (21 aa) + shorter mini-STRC (1076 aa) = 1097 aa
Attempt 1: Most-frequent codons
- GC: 72.3% (TOO HIGH, optimal 40-65%)
- CpG: 221 (67.1/kb, triggers immune response)
- Max homopolymer: 10 nt
Attempt 2: Balanced weighted selection + GC window correction
- GC: 54.5% (optimal range)
- Max GC window (100bp): 65% (acceptable)
- CpG: 120 (36.5/kb) (much better)
- CAI: 0.711 (good, commercial tools can push to 0.85+)
- Max homopolymer: 6 nt (safe)
Files
- Balanced codon-optimized FASTA:
/tmp/strc_final_optimized.fasta - Raw protein FASTA:
/tmp/strc_constructs.fasta - Desktop summary:
~/Desktop/2026-03-19-1319-af3-jobs.md
Note on CAI
0.711 is good but not optimal. Commercial tools (GenSmart, IDT Codon Optimization) use proprietary algorithms that balance CAI, GC, CpG, mRNA secondary structure, splice sites, and restriction sites simultaneously. For a real therapeutic construct, use a commercial optimizer. Our version is a proof-of-concept showing it fits.
AF3 Validation Jobs (pending)
Four constructs generated for AF3 submission to test SP + truncation boundary:
- IgK-SP + shorter mini-STRC (700-1775) — does prepending SP affect fold?
- Residues 650-1775 — wider truncation boundary
- Residues 680-1775 — near the pLDDT dip (res 691 = 31.2)
- Residues 720-1775 — tighter truncation
Job request JSONs on Desktop: ~/Desktop/2026-03-19-1319-af3-jobs.md
Construct Comparison
| Construct | Residues | CDS (bp) | AAV headroom | pTM | Status |
|---|---|---|---|---|---|
| Full STRC | 1-1775 | 5,325 | DOES NOT FIT | 0.63 | Reference |
| Mini-STRC (conservative) | 616-1775 | 3,480 | 1,220 bp | 0.81 | Original |
| Shorter mini-STRC | 700-1775 | 3,228 | 1,472 bp | 0.86 | Recommended |
| C-term only | 1075-1775 | 2,103 | 2,597 bp | 0.87 | Aggressive backup |
Connections
[applies]STRC Mini-STRC Single-Vector Hypothesis — this is the actual vector design- STRC Signal Peptide Validation — IgK SP is part of the construct
- STRC GPI-Anchor Analysis — C-terminal must be intact
- STRC pLDDT Profile and Cut Point Analysis — residue 700 cut point justified by pLDDT data
[see-also]Sonogenetic STRC Computational Proof — alternative promoter design (6xNFAT instead of B8)[about]Misha[see-also]Functional Core Extraction — truncating the disordered N-terminal is a molecular biology instance of this pattern: the inert wrapper (38% disordered, pTM 0.27) was preventing the simple solution (AAV packaging) from working- Logo Strip Normalization — same logic in CSS: transparent canvas is the inert wrapper; ink-trim reveals the functional core, enabling the simple height solution
[part-of]STRC B8 Enhancer Selection — B8 is the OHC-specific enhancer selected for this construct