STRC Research

STRC h03 B8 Enhancer Bp Resolved 2026-04-24

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STRC h03 — B8 Enhancer Bp Resolved (2026-04-24)

Closes the final h03 clinical-vector blocker opened by STRC h03 Clinical-Vector Blockers 2026-04-24 and carried into the 2026-04-25 lit-audit batch. Unblocks Ultra-Mini gBlock order.

Decision

B8_ENHANCER_SIZE_BP = 706 (was 587).

  • Provenance: Zhao et al. 2025, Neuron 113(10):1579–1596 (PMID 40262614). Main text p.1587 + Fig 6A defines B8 composition as E1P3×2 + E2P2×2 + E2P3×2. Module sizes (p.1586, Fig S6A/B): E1P3=93 bp, E2P2=132 bp, E2P3=128 bp. Contiguous back-calc: 2·93 + 2·132 + 2·128 = 706 bp.
  • Prior 587 bp has no primary citation anywhere in the vault or literature. Retracted per AGENTS §0c.4 (never fabricate values).
  • Table S2 (SI Excel) remains the authoritative source for the exact cloned sequence including KpnI/XbaI restriction-site linkers (Key Resources Table). Cloning linkers typically add ≤40 bp; not yet retrieved. Flagged in both scripts as “verify before GMP cloning” — this is a quality-control gate on the order form, not a blocker on design conclusions.

Scripts updated

Both in ~/STRC/models/:

  • ultra_mini_promoter_shortlist.py — B8 row updated to 706 bp with Zhao 2025 provenance; stale output path ~/Brain/research/strc/models/ fixed to Path(__file__).with_suffix(".json") (post-vault-split cleanup).
  • ultramini_vector_cpg_audit.py — B8_ENHANCER_SIZE_BP = 706; stale output and FASTA-input Brain paths fixed.

Re-run output (2026-04-24)

ultra_mini_promoter_shortlist.py:

01_B8_alone_baseline            706 bp,  spare=1138 bp,  tier=3
02_B8_plus_WPRE3                953 bp,  spare= 891 bp,  tier=4  ← top
03_B8_plus_WPRE_full           1299 bp,  spare= 545 bp,  tier=4
04_Myo15_956_alone              956 bp,  spare= 888 bp,  tier=3
05_Myo15_1611_full             1611 bp,  spare= 233 bp,  tier=3
06_B8_plus_Myo15_core_plus_WPRE3 1253 bp, spare= 591 bp, tier=3
07_Prestin_native_plus_WPRE3   2047 bp,  spare=-203 bp,  tier=1  ← overflow

ultramini_vector_cpg_audit.py (full vector with B8 = 706 bp):

Total vector: 3565/4700 bp (75.9% of AAV capacity)
Total CpG:    48  (1.39× human genome 9.7/kb)
B8 contribution: 706 bp, 5 CpG (10.4% of CpG burden)

All design conclusions hold. 119 bp correction does not threaten AAV fit. Top candidate remains 02_B8_plus_WPRE3 with 891 bp spare for future regulatory additions.

Ranking delta

  • h03 tier A → S. Last open clinical-vector blocker closed. lit_audit: partial → fixed. next_step changes from “resolve B8 bp” to “order Ultra-Mini gBlock + B8+WPRE3 cassette; Phase 4 HEK coIP” (when the decision to order is made).
  • Hub frontmatter updated to reflect new tier, lit_audit: fixed, and the lit_audit_note on Table S2 verification as a cloning gate.
  • Per-hypothesis log.md prepended with this closure.
  • Vault log.md entry extended under the 2026-04-24 honest-reset block.

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