Scoville Splice · ScovilleSplice.farm

SS-0092 — Venom Protocol

Pepper Heat Ladder Pepper · C. chinense · 8M SHU

Product Specification

Product ID: SS-0092
Product Name: Venom Protocol
Target Organism: Pepper (C. chinense)
Engineering Tier: Pepper
Series: Heat Ladder
Target SHU: 8,000,000
Pun1/CS Variant: Triple
Gene Count: 9
Construct Length: 14,655 bp
Construct Type: T-DNA binary vector
Selection Marker: nptII (kanamycin resistance)
Promoter System: 2A11
Codon Optimization: pepper
Capsaicinoid Profile: extreme multi
Burn Character: Heat Ladder: Venom Protocol

Gene Cassette Map

LB — [nptII] — [pAMT] — [Pun1/CS Triple] — [BCAT] — [KAS] — [FAR] — [PAL] — [C4H] — [4CL] — [HCT] — RB

Gene	Function	Role
pAMT	Putative aminotransferase	Vanillylamine biosynthesis
Pun1/CS (Triple)	Capsaicin synthase	Acyl-vanillylamine condensation
BCAT	Pathway enzyme	Capsaicinoid pathway
KAS	Pathway enzyme	Capsaicinoid pathway
FAR	Pathway enzyme	Capsaicinoid pathway
PAL	Pathway enzyme	Capsaicinoid pathway
C4H	Pathway enzyme	Capsaicinoid pathway
4CL	Pathway enzyme	Capsaicinoid pathway
HCT	Pathway enzyme	Capsaicinoid pathway

Flux Balance Analysis

Predicted Capsaicin: 300.0 μmol/g
Predicted DHC: 120.0 μmol/g
Total Flux: 3.3546 μmol/h
Vanillylamine Branch: 0.67
Acyl Branch: 0.779
Condensation Efficiency: 0.915
Growth Impact: 0.547 (fraction of WT)
Feasibility: EXPLORATORY

CRISPR Guide RNAs (POX Knockout)

Target	Spacer (20-mer)	PAM	GC%	Efficiency
CaPOX1	AATGCGCCAGCCGCGCTTTT	NGG	60.0%	0.594
CaPOX2	AGTGCGATGTATACAAGTCC	NGG	45.0%	0.676
CaPOX3	ACCCACGTACCCTGCAAGCA	NGG	60.0%	0.693
CaPOX4	CACTCGCAAGTAAATATACC	NGG	40.0%	0.698
CaPOX5	TGCAAACTGGAACCGGATGG	NGG	55.0%	0.684

Transformation Protocol Summary

Vector: T-DNA binary vector in Agrobacterium tumefaciens GV3101
Selection: 50 mg/L kanamycin
Confirmation: PCR with construct-specific primers + HPLC capsaicinoid assay
Expected Timeline: 4-6 weeks (callus to T0 plant)