I wish to knock out certain genes in bacteria by double crossover. For transformation, I am planning to use linear DNA containing antibiotic reistance gene flanked by the region homologous to the gene to be knocked out. I have following doubts.
1) Is it neccessary that the antibiotic reisistance gene be cloned in-frame with the 5' fragment of the target gene? I think it's not.
2) As far as I know, I just need to include cds of the antibiotic resistance gene and it will be expressed using the target genes promoter. Please correct me if I am wrong in stating this.
3) Is it neccessary that the 5' flanking fragment of the target gene on the DNA to be trasformed should be as small as possible? So that the start codon of antibiotic resistance genes is closer to the target gene's promoter and is also easilty recognized during translation?