DNA

Part:BBa_K3602013:Design

Designed by: Flora Fuglsang   Group: iGEM20_SDU-Denmark   (2020-10-23)


rs16901979-C Target Cas12a

The gene sequence itself was looked up on dbSNP, and adjacent nucleotides from the SNP in both the 5’ and 3’ end was added as a “buffer” zone for the Cas12a to bind and to ensure that there is a PAM sequence available so cas12a can be activated by the sequence.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 44
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 167
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 44
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 44
  • 1000
    COMPATIBLE WITH RFC[1000]



Design Notes

The T7 promoter was chosen from neb.com for high yield RNA synthesis. The gene sequence itself was looked up on dbSNP. When the gene was identified, we looked for the PAM sequence specific for Cas12a which is TTTn. Here we found the TTTA PAM sequence and choose the next 24 nucleotides as our target. Then the 88 nucleotides on both sides of the target were included and the whole sequence was ordered from IDT as a gBlock.

Source

The rs16901979 is taken from SNPedia https://www.snpedia.com/index.php/Rs6983267) and the T7 promoter is from neb.com (https://international.neb.com/protocols/2015/11/24/sgrna-synthesis-using-the-hiscribe-quick-t7-high-yield-rna-synthesis-kit-neb-e2050).

References