SBB09 Construct8

Circularly Permutated OmpG- loop 2 break

CPG_L2 N-terminus: PCR Ojb001F/Ojb001R on E. coli MG1655 genomic DNA           (211bp, EcoRI/BamHI) Sub into pBca9145-Bca1144#5                                                       (EcoRI/BamHI, 2057+910, L) Product is pBca9145-M10006           {<N.CPG_L2!} --- Ojb001F                Forward construction of CPG_L2 N term part ctctgGAATTCatgAGATCTgaggaaaggaacgactgg Ojb001R             Reverse construction of CPG_L2 N term part catgtGGATCCttacgcgctataatctaccggcc

CPG_L2 C-terminus: PCR Ojb002F/Ojb003R on E. coli MG1655 genomic DNA        (371 bp, gp=A) PCR Ojb003F/Ojb002R on E. coli MG1655 genomic DNA        (343 bp, gp=B) - PCR Obj002F/Ojb002R on A+B                     (694 bp, EcoRI/BamHI) Digest pBca9145-Bca1144#5                       (EcoRI/BamHI, 2057+910, L) Product is pBca9145-M10007            {} - Ojb002F        Forward construction of CPG_L2 C term part            ctctgGAATTCatgAGATCTggtaaacgtggaacgtggtt Ojb003F         Forward removal of XhoI site in CPG_L2                   actattatctTgagcgcggc Ojb003R         Reverse removal of XhoI site in CPG_L2                   gccgcgctcAagataatagt Ojb002R         Reverse construction of CPG_L2 C term part           catgtGGATCCgaacgagtaatttacgccga

JCA: I made slight mods to nomenclature below. Parts referred to Bjb parts. CPG_L2 complete part: PCR Ojb004F/Ojb001R on pBca9145-M10006      (209 bp, =fragA) PCR Ojb002F/Ojb004R on pBca9145-M10007      (701 bp, =fragB) PCR Ojb002F/Ojb001R on fragA + fragB             (892 bp, EcoRI/BamHI) Sub into pBca9495KC-Bca1144#5                          (EcoRI/BamHI, 2974+910,L) Product is pBca9495KC-M10008           {<CPG_L2!} --- Ojb004F       Forward SOEing oligo for CPG_L2           GGATCTAAGTCTCGTCGCgaggaaaggaacgactggca Ojb004R        Reverse SOEing oligo for CPG_L2           GCGACGAGACTTAGATCCgaacgagtaatttacgccga