SBB09 Construct8
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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!}
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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)
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PCR Obj002F/Ojb002R on A+B (694 bp, EcoRI/BamHI)
Digest pBca9145-Bca1144#5 (EcoRI/BamHI, 2057+910, L)
Product is pBca9145-M10007 {<C.CPG_L2>}
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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!}
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Ojb004F Forward SOEing oligo for CPG_L2 GGATCTAAGTCTCGTCGCgaggaaaggaacgactggca
Ojb004R Reverse SOEing oligo for CPG_L2 GCGACGAGACTTAGATCCgaacgagtaatttacgccga
