Cool work! Have you tried Gateway for library assembly? You could add attB sequences instead of Gibson homology sequences, although you'll need to modify your plasmid to be compatible. Gateway (or the MegaGate variant) has worked for me to clone libraries.
Of course if you're happy with USER then you can just keep using that.
We have not, and it's a good suggestion. Our current workflow is built around the USER approach, but if we run into a roadblock in further optimization then we'll keep it in mind for further optimization.
Cool work! Have you tried Gateway for library assembly? You could add attB sequences instead of Gibson homology sequences, although you'll need to modify your plasmid to be compatible. Gateway (or the MegaGate variant) has worked for me to clone libraries.
Of course if you're happy with USER then you can just keep using that.
We have not, and it's a good suggestion. Our current workflow is built around the USER approach, but if we run into a roadblock in further optimization then we'll keep it in mind for further optimization.
Which organism to source genes from? I would suggest Cryptococcus neoformans fungus. It thrived in the Chernobyl wastelands, it loves gamma-radiation.
https://pubmed.ncbi.nlm.nih.gov/27899501/