Figure 1.

(A) The physical structure of the modified mini-mariner transposon mini-MAR366. The left and right mariner inverted repeats (IR-L and IR-R) flank the pac gene, which encodes resistance to puromycin in Methanosarcina, the E. coli plasmid R6K origin of replication, and the aph gene, which encodes resistance to kanamycin in E. coli. Transcription of the pac gene in Methanosarcina is from the M. voltae methyl reductase operon promoter, pmcr. The transcription terminator tmcr is located immediately upstream of the aph gene. Mini-MAR367 (not shown) is identical to mini-MAR366, except that the region between the inverted repeats is in the opposite orientation. (B) The physical structure of the mini-mariner delivery plasmid pWM381. This plasmid replicates in E. coli by virtue of the high-copy plasmid pMB1 replicon, but cannot replicate in Methanosarcina. The gene encoding the mariner transposase, tnp, is expressed from the strongly transcribed pmcrB promoter of M. barkeri. Plasmids pWM379, pWM383, and pWM385 (not shown) are identical to pWM381, except that the pmcrB promoter is replaced by one of three alternate promoters, as described in Materials and Methods. Additional elements described in the text vary with respect to the orientation of the mini-MAR element; some carry mini-MAR367 in place of mini-MAR366.