I am working with a bacteria that has an active restriction-modification (RM) system against a commonly occurring bp sequence, GATC.
This requires me to remove the GATC sequence from the plasmid I am working with, so it will not be digested after transformation. (no commercial methylase available for GATmC)
This is not a problem for ORFs in the plasmid as they accept degenerate codon substitutions in the sequence pretty well.
However, the E.coli replicon of this vector is based on a pBR322 plasmid, which means the origin of replication is based on RNA I and RNA II primer initiation of replication (back to the old molecular textbooks for this stuff).
My question is essentially this:
If I remove/replace GATC sequences occurring within the ori (RNAI and RNAII sequence) will this completely inhibit plasmid replication in E.coli?
Is there a degeneracy in ori which allows bp substitution? If so, for my purposes I would only need to change a single base to remove the RM recognition motif, so are A/T or C/G more tolerated?
I know this is a pretty specific question but someone who knows a lot about relaxed/stringent plasmid replication might just have tried changing up the sequences.
Thank you in advance.