Co-targeting linker upstream of resistance (Linker 5):

Element	Length (bp)
Homology to resistance cassette for Gibson assembly	18–25
Type IIs restriction site (BspQI or BtsI)	6–8
Genomic sequence upstream of the primary CRISPR cut site	50
The desired insert sequence, usually matched to the frame of the homology arms	Variable
Genomic sequence upstream of the primary CRISPR cut site	50
Type IIs restriction site (BspQI or BtsI) directed upstream	6–8
Diagnostic SacI site	6
Type IIs restriction site (BspQI or BtsI) directed downstream	6–8
Genomic sequence upstream of the secondary CRISPR cut site	50
Homology to the bacterial backbone for Gibson assembly	18–25