SIL072006-Construction of pSB1A2-J23017

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