Whether or not stop codons are left will depend upon the specific cassette you are using. The original KD3 and KD4 cassettes do have stop codons in all frames whereas I think KD13 does not. Also many other different cassettes have been made to use, so it depends upon the cassette. However I would not be so worried about it. Many genes in an operon are not translationally coupled, if an ORF still has a good RBS then the upstream translation (or lack thereof) may not have much effect. Likewise transcriptional polarity is not going to be an issue unless you have a long untranslated region.

But you can design your own cassettes so that it leaves behind whatever sequence you wish (plus of course the FRT site).

Michael,

Thanks for your answer, it´s helpful to have a colleague with whom to "bump heads" to work things out.

The operons I´m working with have translational coupling, so I need to keep this in mind. Attached is my take on pKD13 has stop codons in the three phases, either in the priming site, the scar sequence, or the FRT site. Best option may be designing the priming site without a stop codon in phase 3 and do the construct using that phase.

Hi Teca, Happy to help if I can and nice to meet you. We have clearly worked in related areas over the years.

So I'm a little confused to the details of what you are trying to do. Are you hoping to precisely delete one gene that is in the middle of an operon? It is true that the scar has stop codons in all 3 frames, that was intentional. But note that it is only in one direction. If you use it in the other orientation I believe (from the original paper) that it only has a stop in one direction. You could also make mutations to remove these stops. I would also note that KD3 and KD4 are designed to have translational starts in one orientation, so while you would remove translational coupling you could preserve translation of the downstream gene by reinitiation.

I hope this gives you a few things to think about that might solve your problem.

You might also look at some other similar protocols that let you create deletions and such without any scars. There are likely to be dozens of similar procedures but here are two I found very quickly.

Cox, M.M., Layton, S.L., Jiang, T. et al. Scarless and site-directed mutagenesis in Salmonella enteritidischromosome. BMC Biotechnol 7, 59 (2007). https://doi.org/10.1186/1472-6750-7-59

Stringer AM, Singh N, Yermakova A, Petrone BL, Amarasinghe JJ, Reyes-Diaz L, et al. (2012) FRUIT, a Scar-Free System for Targeted Chromosomal Mutagenesis, Epitope Tagging, and Promoter Replacement in Escherichia coli and Salmonella enterica. PLoS ONE 7(9): e44841. https://doi.org/10.1371/journal.pone.0044841