Dear colleagues,
I have designed a construct for eukaryotic expression. Before I submit it for synthesis, I would like to find out about the possible implications of 3 (unintended) features that were listed in the analysis the gene synthesis people sent to me (without explanation, of course, that's why I am asking)
"Short Dyads
TCGACGGCAGCT
CCCAGAAGACCC
Palindromic Sequences
GCCGTCGACGGC"
Despite finding in the internet only remarks that there are features which have these names, are there any caveats, i.e. one should avoid them? May they bind e.g. transcription factors etc, ?. Should I remove them (i.e. do some conservative mutations) ? How many bases would you suggest to change?
Thanks for your suggestions!
I think these sequences are too short to be of concern. Palindromes longer than 100bp have been shown to interfere with plasmid replication in prokaryotes (https://www.jstage.jst.go.jp/article/jgam1955/32/5/32_5_393/_pdf). Palindromes can cause deletions, but a structure with a 20bp stem caused at least 100x lower frequency of deletions than a structure with a 48bp stem (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1204783/pdf/ge1294991.pdf). Another paper reports 34bp as a lower limit to expression inhibition (http://www.ncbi.nlm.nih.gov/pubmed/12557395). A minimum of 4-5bp must also form a loop in any hairpin/cruciform formed, so your sequences would only give you a 4bp stem. If these sequences were known binding sites, they could be of concern...still the duplications are quite close to each other, maybe they would not be capable of engaging something in binding both at the same time (think protein dimer). Check if the search for palindromes that was used allowed for unpaired bases in the central part. If not, you could repeat the search while allowing for a spacer/central loop.
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