Part:BBa_K5055001:Design
NdeI_PDS_insert
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 354
Illegal NgoMIV site found at 725 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
We had trouble synthesizing these sequences because DNA manufacturers are not able to synthesize sequences with repeats over 19 bp. Therefore, we decided to divide our sequences into two: one sequence encoding the sense sequence of our hairpin RNA and one sequence encoding the antisense sequence of our hairpin RNA. To be able to assemble these two parts, we chose our loop sequence so that it contained the restriction site of the enzyme NdeI. The loop structure allows a better detection of the lhRNA by the Dicer complex. It also confers a better stability to the solution, compared to double-stranded RNA. We needed to incorporate a few bases providing a loop configuration between sense and antisense sequences of the chosen targets. To design the loop, we saw in the literature that for lhRNA designs, we should use loop sequences of around 8 nucleotides for a final size of around 1kb, with a C and a G base at the extremities of the loop for increased stability. It then requires a random mismatch for the loop opening.
Source
This part corresponds to a fragment of the phytoene desaturase of Beta vulgaris. Its sequence was found on NCBI (NCBI Reference Sequence: XM_048641752.2).