Part:BBa_K4812019:Design

In designing this part, several design considerations were taken into account. First, ensuring that the primer is between 25 and 45 bases in length, with the desired mutation being in the middle of the sequence (at least 10 bases from either ends). Additionally, ensuring that the primer’s melting temperature (Tm) is ≥78°C, and that both the forward and reverse primers have similar melting points. Third, ensuring that the GC content is ≥40% and that the primer terminates in a G or C on its 3’ end. Lastly, ensuring that the number of mismatches between the primer and the target sequence is ≤3 bases in a row.