Protein coding linker downstream of resistance (Linker 4):

Element	Length (bp)
Homology to resistance cassette for Gibson assembly	18–25
The complete 5′ UTR of the endogenous gene, plus ∼300 bp extra on the 5′ end to include remote promoters. Alternatively, a different, known promoter can be used.	Variable
A start codon followed by the coding sequence of the desired fusion tag. Adding introns is highly recommended.	Variable
Linker peptide and/or epitope tag (Optional but recommended)	Variable
The genomic sequence from the endogenous start codon to the CRISPR cut site, mutated to not include the gRNA sequence	Variable
Genomic sequence downstream of the CRISPR cut site	50
Type IIs restriction site (BspQI or BtsI)	6–8
Diagnostic SacI site	6
Artificial primer binding site	18–30
Homology to the bacterial backbone for Gibson assembly	18–25