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5kb 5 arm was designed to be inserted into the swai

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A 3.5Kb 5’ arm was designed to be inserted into the SwaI site and a 3.0Kb 3’ arm was designed to be inserted into the XhoI site. There was considerable difficulty in cloning the 5’ arm into the SwaI site, thus a shorter 5’ arm at 3.1Kb was designed but to no avail. The 5’ arm must be inserted before the 3’ arm. Then a polylinker was designed to be first inserted into the SwaI site, after which the 5’ arm will be subsequently cloned into the ploxPFlpneo plasmid utilizing one of the restriction sites in the “SwaI polylinker.” The SwaI polylinker sequence is shown below in capital letters: agacgcATTTAAATTCGAATTCCTCAGCAGATCTGGGCCCATTTAAATagacgc In the Linker: BbvC I = CC*TCAGC Bgl II = A*GATCT BstB I = TT*CGAA EcoR I = G*AATTC Apa I = GGGCC*C Swa I = ATTT*AAAT Outside the Linker: Swa I = ATTT*AAAT BamH I = G*GATCC Apa I = GGGCC*C Xho I = C*TCGAG * denotes where the restriction site is cut by the restriction enzyme.
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