SIL072006-Construction of pSB1A2-J23017

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Rlambda plasmid with traG knockout
PCR JL20/JL21 on Rlambda gen. (2268 bp, MfeI/BglII) Sub into pGLN (BamHI/EcoRI) Product is pJL20(rename)

IPCR JL22/JL23 on pJL20(rename) (3454 bp, EcoRI/PstI) Sub in pSB1A2-Bca9007 (1015 bp, EcoRI/PstI) Product is pJL22(rename)

PCR JL18/JL19 on pJL22(rename) for D/W

JL20 Forward BglII to BamHI for traG-5' knockout cassette 

GAGTCAGATCTgattatgttggtgcgctg

JL21 Reverse MfeI to EcoRI for traG-5' knockout cassette 

gctggCAATTGcattcgatttccagtacaggc

JL22 Reverse EcoRI IPCR of traG gctgcGAATTCgatactggaaggaatgggc JL23 Forward PstI IPCR of traG cattgCTGCAGacggcagcattgaagaggcg

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