SBB09 Construct9

Circularly Permutated OmpG- Loop 6 Break

CPG_L6 N-terminus:

PCR Ojb005F/Ojb003R on E. coli MG1655 genomic DNA        (548 bp, gp=A) PCR Ojb003F/Ojb005R on E. coli MG1655 genomic DNA        (175 bp, gp=B) - PCR Obj005F/Ojb005R on A+B                     (703 bp, EcoRI/BamHI) Digest pBca9145-Bca1144#5                      (EcoRI/BamHI, 2057+910, L) Product is pBca9145-M10009            {<N.CPG_L6!} - Ojb005F         Forward construction of CPG_L6 N term part          ctctgGAATTCatgAGATCTgaggaaaggaacgactgg Ojb003F         Forward removal of XhoI site in CPG_L6     actattatctTgagcgcggc Ojb003R         Reverse removal of XhoI site in CPG_L6          gccgcgctcAagataatagt Ojb005R         Reverse construction of CPG_L6 N term part     catgtGGATCCttactgccagtcccagttactcc

JCA note: When you gel purify the Ojb003F/Ojb005R and Ojb006F/Ojb006R PCRs, you should add 250uL of isopropanol to the melted ADB cocktail before passing it through the column due to the short size

JCA note: below it said "L2"; I changed it to "L6". Also note that you should add isopropanol during the gel purification for Ojb006F/Ojb004R JCA: Ojb002R and Ojb006R are the same. Ojb006R was not ordered.

CPG_L6 C-terminus:

PCR Ojb006F/Ojb002R on E. coli MG1655 genomic DNA           (202 bp, EcoRI/BamHI) Sub into pBca9145-Bca1144#5                                 (EcoRI/BamHI, 2057+910, L) Product is pBca9145-M10010           {} --- Ojb006F             Forward construction of CPG_L2 C term part        ctctgGAATTCatgAGATCTgatgatattgaacgtgaagg Ojb006R             Reverse construction of CPG_L2 C term part       catgtGGATCCgaacgagtaatttacgccga Ojb002R             Reverse construction of CPG_L2 C term part           catgtGGATCCgaacgagtaatttacgccga

Complete Part Construction File:

JCA note: below it referred to cBjb numbers, I changed them to M numbers PCR Ojb004F/Ojb005R on pBca9145-M10009     (701 bp, =fragA) PCR Ojb006F/Ojb004R on pBca9145-M10010     (209 bp, =fragB) PCR Ojb006F/Ojb005R on fragA + fragB        (892 bp, EcoRI/BamHI) Sub into pBca9495KC-Bca1144#5                 (EcoRI/BamHI, 2974+910, L) Product is pBca9495KC-M10011            {<CPG_L6!} Ojb004F    Forward SOEing oligo for CPG      GGATCTAAGTCTCGTCGCgaggaaaggaacgactggca Ojb004R      Reverse SOEing oligo for CPG             GCGACGAGACTTAGATCCgaacgagtaatttacgccga