Composite

Part:BBa_K5471011:Design

Designed by: Michail Diamantakis   Group: iGEM24_Patras-Med   (2024-09-29)

EF1a promoter infused with mir-195


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 452
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 452
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 600
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 452
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 452
    Illegal NgoMIV site found at 335
    Illegal AgeI site found at 60
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1105


Design Notes

This new composite part was carefully designed by our team to simulate the intragenic expression of most microRNAs, by imitating the genomic locus in which the hsa-mir-195 gene (87bp) is located. The logic behind it is inspired by the construct "sc AAV miR26a eGFP" used in the publication cited in "Sources"

The genomic locus chr17:7017465:7017851 (387bp),(https://parts.igem.org/Part:BBa_K5471009), can be treated as the “gene of interest” that is inserted in an already existing cassette or plasmid vector. In our case, the 150bp extra sequences flanking the hsa-mir-195 gene on either side, are identical to the ones that flank the gene naturally in the human genome (150+150+87=387).

The goal of this faithful imitation of this human genomic locus is to drive the natural transcription and modification processes so that the mature hsa-mir-195-5p is produced inside the treated cells. This method promises a stronger expression of the mature microRNA, resulting in bigger numbers of it, compared to other methods that insert the mature miRNA directly or use shRNA mimics.

Specifically, the EF1a promoter used is (https://parts.igem.org/Part:BBa_K5471005) . Since we aim to insert a 387bp length sequence, we decided it was crucial to preserve the parts of the intronic sequence near its 5’ and 3’ end, and to preserve the original length of the intron. With these principles in mind, we decided that our “gene of interest” will be inserted in the position 334 of the original EF1a promoter. The combined length of this new part is 1178 bp, which is longer than the original EF1a promoter, but the same size as many EF1a promoters that are commonly used. At the same time, the sequences of the EF1a intron close to the 5’ and 3’ ends remained untouched, so that the natural excision of the intron from cellular mechanisms can take place.


Source

This part was designed by us, taking inspiration from "sc AAV miR26a eGFP" https://www.addgene.org/21894/ sc AAV miR26a eGFP was a gift from Joshua Mendell (Addgene plasmid # 21894 ; http://n2t.net/addgene:21894 ; RRID:Addgene_21894)


References

Kota J, Chivukula RR, O'Donnell KA, Wentzel EA, Montgomery CL, Hwang HW, Chang TC, Vivekanandan P, Torbenson M, Clark KR, Mendell JR, Mendell JT. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell. 2009 Jun 12;137(6):1005-17. doi: 10.1016/j.cell.2009.04.021. PMID: 19524505; PMCID: PMC2722880.