1. MiRNA - Limited to hsa and mmu MiRNAs with clear loop sequences

2. PRECURSOR NUMBER - A total of 1820 precursors, including 951 for hsa and 868 for mmu.

3. INFO REQUIRED - Customers are required to provide the following information: Target genes - sequence, name, species; miRNA - name, species.

4. TARGET ADJUSTMENT - The target will be either extended or truncated based on the MiRNA mature sequence. Adjustment will also be made based on Bulges in the natural MiRNA structure to ensure that the final secondary structure matches or closely resembles the natural MiRNA.

5. ALGORITHM LIMITATIONS - Due to algorithm limitations, the final secondary structure may have slight differences from natural MiRNA, but this does not affect the functionality of shRNA.

6. PRIMARY SEQUENCE - The final miRNA (primary) will be generated and provided in .GB format for download. It can be placed between the Pol II promoter and PolyA. If necessary, restriction enzyme sites can be introduced without affecting the functionality of miRNA/shRNA.

7. MATURE SEQUENCES - Two mature sequences (5P and 3P) will be generated. The 5P corresponds to ANTISENSE and should only target the gene of interest, while the 3P should not target any mRNA of the target species.

8. OFF-TARGET PREDICTION - Two modes are provided, namely STRICT MODE and LOOSE MODE. Report is based on STRICT MODE, while results from LOOSE MODE are for reference only.