Fig. 3.
Schematic representation of the integration of pMM656_ldhnP into the genome of P. vulgatus. The promoter PRhaKIPAO and the coding gene of NanoLuc of pMM656 were replaced with the ldh gene (BVU_2499) of P. vulgatus including 250 bp of the upstream region. The tyrosine integrase NBU2 Int catalyzed the stable integration of pMM656_ldhnP into one of two attN2 sites in the P. vulgatus genome. The two attN2 sites are located at the 3′ ends of tRNASer genes (BVU_2451 at nt 3,152,564 and BVU_2094 at nt 2,710,348, respectively). AmpR, ampicillin resistance cassette; ErmR, erythromycin resistance cassette; RP4, origin of transfer; R6K, origin of replication. Black horizontal arrows indicate the position of the primer pair attB2451_for/attB2094_for and attB_inErm_rev. The primers were used to generate a DNA fragment for sequencing and to determine the position of integration