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!}
-----------------------------------------------------------
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>}
-------------------------------------------------------------
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
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