Part:BBa_K5232004

Usage and Biology

It has a sequence that is fully complementary to primer3 [1], with the middle portion having a recognition sequence for the Cas3-Cascade/crRNA complex.

Figure 1.
Cas3 recognizes a double-stranded PAM sequence and a single-stranded spacer sequence for activation. It has been reported by Yoshimi et al. that even partial PAM sequences combined with a complete spacer sequence can still elicit some level of activity [2]. Therefore, a sequence complementary to the spacer sequence was incorporated into the ds-template.In other words, the ds-template sequentially contains a complementary sequence to the primer, a PAM sequence, and one strand of the complementary spacer sequence, starting from the 3' end.

Figure 2.
When ds Amp and Cas3 system were connected, no increase in NC fluorescence intensity was observed, and the graph showed a slope dependent on target concentration.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Komiya, K., Noda, C. & Yamamura, M. (2024). Characterization of Cascaded DNA Generation Reaction for Amplifying DNA Signal.New Gener. Comput. 42, 237-252. https://doi.org/10.1007/s00354-024-00249-2

Yoshimi, K., Takeshita, K., Kodera, N., Shibumura, S., Yamauchi, Y., Omatsu, M., ... & Mashimo, T. (2022). Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3. Nature Communications, 13(4917), 1-11. https://doi.org/10.1038/s41467-022-32618-0