Figure 3.

Schematic representation of two successive allelic replacements using SeqDelTA. The LHR is conserved during the deletion process, allowing the recycling of the resistance markers at each step. The RHR is redesigned at each step to inactivate the following TA module and delete the cassette cargo in between. The inactivation of the TA system is performed by knocking the toxin but leaving the antitoxin intact. A total of 18 sequential replacements were performed. The last deletion step was carried out using a suicide plasmid.