In E. coli and related species you can use the Lambda-Red system developed by Datsenko & Wanner to generate allelic recombination, with just 20 bp homology regions in each end of your PCR product. Wondering if there was something similar for Gram positives?
Yes... long flanking homoly (LFH-PCR)
http://www.sciencedirect.com/science/article/pii/0378111995006524
http://www.dnai.org/media/bioinformatics/ccli/CD/readings/wach1996.pdf
In E. coli recombineering has also been developed by Don Court. In mycobacteriophage Graham Hatfull found some analogous genes that work in a recombineering system in mycobacteria.
There is a system available from Sigma Aldrich ...called Targetron ...they claim for instance B. subtilis.
To be honest ...never tried it so far!
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