shRNA Target Design
Design shRNA sequences with knockdown efficiency prediction
shRNA / siRNA Design Lab
Automated knockdown candidate designer combining Reynolds, Ui-Tei, and thermodynamic asymmetry
Design Settings
U6 requires starting 'G'. H1 is flexible.
Ready to Design
Enter an mRNA sequence on the left and run the analysis.
Tool Guide
Definition
shRNA target design tool — Extracts 19–22 nt RNAi target candidates from a target mRNA, then evaluates Reynolds rules, thermodynamic asymmetry (AU-rich 5' seed), GC%, and secondary structure to predict knockdown efficiency. Outputs in shRNA stem-loop format (e.g., TRC pLKO.1).
Purpose
(1) Build stable knockdown cell lines for loss-of-function studies (pLKO.1 lentivirus) (2) Compare 3–5 candidates per gene and pick the highest-efficiency hit (3) Design matching scramble controls (4) Cassettes compatible with inducible (Tet-on/off) knockdown systems
How to Use
① Enter target mRNA sequence (RefSeq NM_xxxxx or paste) ② Choose candidate length (19, 21, 22 nt) ③ Loop sequence (TRC: CTCGAG, miR-30: native backbone) ④ Output: • Sense/antisense candidates • Full shRNA hairpin (sense-loop-antisense-poly-T) • Predicted efficiency score • Annealed oligos with 5'/3' overhangs for synthesis (Currently coming-soon)
Examples
Example) shRNA for human GAPDH knockdown → Input: GAPDH mRNA (NM_002046) → Candidate 1: 5'-GAAGGTGAAGGTCGGAGTC-3' (19 nt, predicted score 8/10) → Hairpin (TRC): 5'-CCGG[sense]CTCGAG[antisense]TTTTT-3' → Ligate into pLKO.1 (AgeI/EcoRI cut) → HEK293 transduction + puromycin selection → ~80% GAPDH knockdown