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. 2006 Mar;72(3):1878–1885. doi: 10.1128/AEM.72.3.1878-1885.2006

FIG. 1.

FIG. 1.

Construction of pFNLTP16 derivatives containing modified and unmodified pMiniHimar to optimize transposase and kanamycin resistance. The pFNLTP16 delivery vehicle was derived from pFNLTP9 (37), a temperature-sensitive derivative of pFNLTP1. Unique restriction sites (multiple cloning site 2 [MCS2]; NdeI, EcoRI, SmaI, NotI, NheI, and XhoI between the KpnI and BamHI sites) were added to pFNLTP9 to generate pFNLTP11. Inverse PCR was performed on pFNLTP11 to generate pFNLTP16, deleting npt. The pUC ori and β-lactamase gene (bla) encoding ampicillin resistance are from pCR2.1-TOPO (light gray arrow or box) (Invitrogen), while repA, orf2, and orf3 are from pFNL10 (dark gray arrows) (46). The pFNLTP16 vehicle was combined with unmodified pMiniHimar using NotI to generate pFNLTP16 H1. The transposase (tnp; striped arrow) is provided in cis with (pFNLTP16 H2 and H4) or without (pFNLTP16 H1 and H3) the addition of the acpA promoter (pacp; striped small box) upstream at PvuI. Himar1, shown in white between the inverted repeats (IR), contains npt and the R6K origin. Expression of npt in pFNLTP16 H3 and H4 is under the control of the F. tularensis LVS groEL promoter (pgro).