JL081406-Construction of J23067,J23068,J23069 for traG knockout

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PCR JL20/JL21 on RP4 gen. (2268 bp, MfeI/BglII)
Sub into pGLN (BamHI/EcoRI)
Product is pJL20(J23067)


IPCR JL22/JL23 on pJL20(J23067) (3454 bp, EcoRI/PstI)
Sub in pSB1A2-Bca9007 (1015 bp, EcoRI/PstI)
Product is pJL22(J23068)


PCR JL18/JL19 on pJL22(J23068) for D/W (bp, DpnI)
Datsenko Wanner on pKD46/Rlambda,
Product is J23069


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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