Variables that need to be considered. Similar Recognition Sequences, and their use in gene manipulation. Bgl II and Bam HI Ligation. It would be desirable that the vector could be easily transferred into recipient strain by one of the previously mentioned methods of gene transfer. The use of E. coli as the definitive host for genetic manipulations has definitely biased the choice of vectors and choice of gene transfer.
Origin of replication is on the opposite side of plasmid from the two drug resistance genes.
Go through and understand at least one cloning strategy, using Bam HI site in Tc r gene. It would thus considerably increase the size of the vector. However, codons always read in threes. Code is Degenerate. Mutations will and do arise naturally and can also occur with a little help from mutagens.