Part:BBa_K3893001:Design

dsRNA designed for the target gene Velvet

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 170
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

For the design of the dsRNA, we used the E-RNAi program and we validated its physicochemical characteristics [1]. As criteria, we selected 21 nucleotide target sequences that begin with AA and are located within a region of the coding sequence, 50-100 nucleotides of the AUG start codon, and within 50-100 nucleotides of the stop codon. The presence of AA at the beginning of the sequence allows the use of dTdT at the 3 'end of the antisense sequence. We considered that the GC content should be less than 50% and we avoided sequences with repeats of three or more G or C, since its presence initiates intramolecular secondary structures that prevent silencing hybridization. Furthermore, we confirmed that RFC10-compatible restriction sites are not found in the sequences. Subsequently, we analyzed the homology with other species, to ensure the specificity of the designed dsRNAs and thus maintain biosafety and biosecurity of environmental organisms [2].

Source

The base sequence was the coding sequence of the gene, taken from NCBI; which was subsequently processed to design the dsRNA.

References

[1] H. Thomas, and M. Boutros. “E-RNAi: a web application for the multi-species design of RNAi reagents--2010 update.” Nucleic acids research vol. 38,Web Server issue (2010): W332-9. doi:10.1093/nar/gkq317

[2] Fletcher, S. J., Reeves, P. T., Hoang, B. T., & Mitter, N. (2020). A perspective on RNAi-based biopesticides. Frontiers in plant science, 11, 51.