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
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