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Design and Synthesis of the miniTn7BB-Gm

As a first step to undertake the construction of the miniTn7 biobrick toolkit, we designed a synthetic miniTn7 minitransposon from which all derivatives could be easily generated. The design was inspired on the miniTn7 transposon set constructed by Choi et al. (2005). The main features of this initial construct, designated miniTn7BB-Gm are: (i) wild-type Tn7R and Tn7L ends, enabling transposition in vivo when the Tn7 transposase is expressed in trans, (ii) prefix and suffix with unique EcoRI, XbaI, SpeI and PstI restriction sites, fully compatible with Assembly Standard 10, (iii) annealing sequences for standard primers to facilitate amplification and sequencing of cloned BioBricks, (iv) transcriptional terminators flanking the multi-cloning site to efficiently insulate cloned BioBricks from vector transcription, (v) a selectable gentamycin (Gm) resistance cassette flanked by duplicated SphI and NcoI sites to facilitate marker replacement, (vi) flipase recognition elements (FRTs) flanking the gentamycin resistance cassette, to allow clean excision of the marker in strains bearing transposon insertions, and (vii) SfiI sites flanking the complete transposon to facilitate cloning into the appropriate plasmid vectors. A schematic of miniTn7BB-Gm showcasing the aforementioned features is shown in Figure 1. The miniTn7BB-Gm transposon was designed by David Caballero and commercially synthesized (Mr. Gene)

Figure 1. Schematic of the miniTn7BB-Gm minitransposon. Transposon ends (red boxes), relevant restriction sites (vertical lines), FRT elements (blue circles), transcriptional terminators (red hexagons), Gm resistance (green box), BioBrick cloning site (cyan box) and primer annealing targets (blue arrows) are indicated