I want to knock out a gene in a Streptococcus bacterial strain and I'd like to use allelic exchange method.
I studied in an article that it is better to use an antibiotic resistance gene without a promoter that is flanked by a homology arm on each side of the target gene.
I chose two homology arms ( each about 800 bp one of them start from the translation start codon and another end with translation stop codon of the target gene ).
my antibiotic resistance gene is about 800 bp and I only inserted its ORF between these two homology arms.
my question is that do I need to add any other sequence that my antibiotic resistance gene need to be translated by the ribosome or the ORF is enough?
I think because I don't want to delete the promoter and the terminator sequences of the target gene, the antibiotic resistance gene can be transcribed by them but I don't know if I need other sequences to add between homology arm and the ORF of the antibiotic resistance gene?
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