Part:BBa_M31511
This is a refactored M13K07 genome. This is only the section between the unique HpaI site in Gene II and the unique BamHI site in Gene III. This part was designed to remove as much overlap as possible between genetic elements so that individual genes could be modified without worrying about affecting other sections of the genome.
I attempted to remove all overlapping sites in this section of the genome. What I did was copy the overlapping section (usually an RBS and/or promoter overlapping the previous ORF) and repeat it. I changed the wobble positions of every other codon on the ORF’s so that they wouldn’t homologously recombine with the repeated sequence. Additionally, as was done on the T7.1 refactoring, I added two new cut sites between the separated genes. I chose to keep the overlap between the gene VIII promoter and gene IX RBS (and the resulting mixed order of genetic elements) changing that order seemed to be a much more significant change than the others.
| Overlapping Areas | Added Restriction Sites | Modified sequence |
|---|---|---|
| Gene V Promoter/RBS - Gene X ORF | AflII (CTTAAG); ApaI (GGGCCC) | ccaacgAcctgaTtggtaCaatgaAccagtActtaaGatcgcCtaaCTTAA GGGCCCaacgtcctgactggtataatgagccagttcttaaaatcgcataa |
| Gene VII RBS - Gene V ORF | BmtI (GCTAGC); BssHII (GCGCGC) | gttccAgctaaAtaaGCTAGCGCGCgttccggctaagtaa |
| Gene VIII Promoter/IX RBS/ORF – Gene VII ORF | KpnI (GGTACC); NcoI (CCATGG) | aatTtccgtCgtactCtgtttTgcgctCggtatTatcgcCgggggCcaaagGtga GGTACCATGGaatctccgttgtactttgtttcgcgcttggtataatcgctgggggtcaaag |
| Gene VIII RBS/ORF – Gene IX ORF | NheI (GCTAGC); MfeI (CAATTG) | taatGgaaacCtcctcTtgaGCTAGCAATTGtaatggaaacttcctcatga |
| Gene III Promoter – Gene VIII ORF | BclI (TGATCA); BglII (AGATCT) | aattcacTtcgaaGgcaagTtgaTGATCAGATCTaattcacctcgaaagcaagctga |
| None |