Sequencing of the insert of the final construct used to generate the ∆<i>rv</i><i>0191</i> M.tb mutant

Here, the fragment of pGOAL17 containing the<i> sacB </i>and <i>LacZ</i> has been cloned into the p2NIL-0191US/DS construct, and this was sequenced again to check for integrity. Since the insert is large, many sequencing reactions were performed, using different primers, named primer 1 to primer 9. Therefore, alignments were performed with all data, to ensure coverage of the entire region.The last two files are the in silico designed constructs, used as template for screening by restriction enzyme digestion (RED) and for the sequencing alignments. Since cloning the fragment from pGOAL17 could face either direction, since it was a blunt end cloning, there are two possible designs of the final construct.