Part:BBa_K4636005:Design

We obey the standard PCR primer design consideration and used Primer 3[1] to search the suitable site then check it by IDT OligoAnalyzer™ Tool. [2]

Standard PCR primer design consideration :.

(1) Length : 18~24 base pairs.
(2) GC content : 40%~60%.
(3) 3’ end with highly GC content.
(4) Tm : 50~60°C

For restriction site sequence design, we refer to previous study [3], [4], [5]:

(1) Restriction site : To ligate with pUC19 vector, we design restriction site BamH1.
(2) Sitting sequence (4~5 b.p.) : Before restriction site and it’s for enzyme activity. Rule : Avoid the same sequence as RE and have no GG or CC sequence.
(3) Annealing site :Complementary to Insert_0004771 (BBa_K46360041).
(4) Random sequence : Adjust Tm value of primer.

Reference

1. https://primer3.ut.ee/
2. https://sg.idtdna.com/pages/tools/oligoanalyzer
3. https://www.addgene.org/protocols/primer-design/
4. https://international.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
5. https://www.researchgate.net/post/Are_the_sitting_sequences_added_before_the_PCR_primers_arbitrary_or_specific_to_restriction_endonucleases