Davidying: Refactoring the M13 Genome

Modifications
1.	Separate the g2/g10 ORF from the promoter g5/RBS g5.

Original: (Red = g2/g10 ORF, underline = g5 promoter, italics = g5 RBS) c,caa,cgt,cct,gac,tgg,tat,aat,gag,cca,gtt,ctt,aaa,atc,g | ca,taa | ggta | attcaca

a.	Repeat red part (g2/g10 ORF) and disrupt the new coding sequence with silent mutations to disrupt the g5 promoter sequence, and to prevent homologous recombination: c,caa,cgt,cct,gac,tgg,taC,aaC,gaA,ccC,gtA,ctA,aaG,atT,gcG,taaccaacgtcctgactggtataatgagccagttcttaaaatcg | cataaggta | attcaca

b.	Add two overlapping restriction sites (ApaI and AvrII) at the boundary between the two parts. c,caa,cgt,cct,gac,tgg,taC,aaC,gaA,ccC,gtA,ctA,aaG,atT,gcG,taaGGGCC|C|CTAGGccaacgtcctgactggtataatgagccagttcttaaaatcg | cataaggta | attcaca			ApaI	     AvrII

Final sequence: ccaacgtcctgactggtaCaaCgaAccCgtActAaaGatTgcGtaaGGGCCCCTAGGccaacgtcctgactggtataatgagccagttcttaaaatcgcataaggtaattcaca

2.	Separate the g5 ORF from the g7 RBS.

Original: (Red = g5 ORF, underline = g7 RBS) gttccggctaagtaa | c

a.	Repeat red part (g5 ORF) and disrupt the new ORF coding sequence with silent mutations, as above.

gtC,ccT,gcG,aaA,taagttccggctaagtaac

b.	Add two overlapping restriction sites (KasI and MfeI) at the boundary between the two parts. gtC,ccT,gcG,aaA,taaGGCGC|C|AATTGgttccggctaagtaac

Final sequence: gtCccTgcGaaAtaaGGCGCCAATTGgttccggctaagtaac

New Parts

 * BBa_M31337: Project part, contains M13K07 genome from HpaI site to BamHI site
 * BBa_M31338: Coding sequence, contains partial gene 2 (and embedded gene 10) from HpaI site to end of gene, marked by ApaI site.