The alignment file exhibits suboptimal quality; consider adjusting parameters such as -g 0.1,0.9 and -f 0.1. However, obtaining satisfactory results may be challenging due to the poor quality of the multialignment. Additionally, your fasta file appears to be too short, and the conserved region is inadequately small. It might be worth exploring the use of multiPrime2-GUI to search for potential primer candidates, although I cannot assure optimal results.

Dear @guangingmai ,
I regret to inform you that I have attempted to utilize your input file with multiPrime2-GUI, but unfortunately, it has proven unsuccessful. The challenging quality of the multiple alignment file continues to pose difficulties for the primer design process. If there are alternative approaches or additional information that could enhance the alignment quality, please provide them so we can explore other avenues for a successful outcome.