SBB09 Construct9
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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 {<C.CPG_L6>} ----------------------------------------------------------- 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