 genetic elements within a transgene at a single insertion site eliminates the problem of genomic position effects associated with independently introducing variants of transgenes at different loci. Furthermore, adjacent genetic elements could be rearranged at a single locus (e.g., switching the positions of a promoter and transcriptional insulator to test how well the insulator blocks transcription). The ability of Hin recombinase to invert large and small DNA fragments and adjacent flippable elements demonstrates the potential flexibility of the Hin/hix system.
The capability of HinLVA to flip adjacent DNA segments indicates that this system could be scaled up to accommodate more complex pancake stacks. As an application in comparative genomics, flippable DNA segment arrays could serve as a model to improve our understanding of syntenic genome rearrangements that have occurred during evolution. Chromosomal regions exist as syntenic modules arranged in different orders and orientations in the genomes of related species. Each syntenic module can be considered a burnt pancake that has a particular order and orientation. Phylogenetic relationships between species can be inferred by using BPP mathematical modeling to compute the minimum number of rearrangements that link two syntenic genomes (see  for review). Hin-mediated rearrangements of an array containing different sized DNA fragments would help refine the mathematical model by accounting for the impact of differences in sequence composition and lengths of syntenic modules. Using Hin/hix to sort DNA fragment permutations in vivo expands the horizons for the emerging field of applied DNA-based computing.
Construction of parts and plasmids
All genetic elements reported here are standardized parts (prefixed "BBa_" and "pSB") that are flanked by universal "BioBrick" cloning sites  and are documented and distributed by the MIT Registry of Standard Biological Parts .E. coli strain JM109 was used for cloning and as the chassis for our system. The recombinational enhancer (RE, BBa_J3101) , hixC (BBa_J44000) , RBS rev (BBa_J44001) and pBAD rev (BBa_J44002) were assembled from 20 – 60 bp DNA oligomers that were designed using the "Oligo Cuts Optimization Program" . EcoRI (5') and PstI (3') single stranded extensions were manually added to the terminal oligomers. An equimolar mix of single-stranded oligomers in 1× annealing buffer [100 mM NaCl; 10 mM Tris-HCl, pH 7.4] was incubated at 100°C for 5 minutes, then slowly cooled to ambient temperature in a water bath to produce double stranded DNA with EcoRI (5') and PstI (3') single stranded overhangs. The annealed DNA was ligated into linearized (EcoRI and PstI digested) cloning vector pSB1A2 using T4 ligase (Promega). Hin (BBa_J31000), HinLVA (BBa_J31001), and tetA(C) (BBa_J31007), and were cloned by polymerase chain reaction (PCR). Standard BioBrick prefix cloning sites (EcoRI, NotI and XbaI) or suffix cloning sites (SpeI, NotI and PstI) were included in the forward or reverse PCR primer, respectively. PCR amplicons were digested with EcoRI and PstI, electrophoresed in an agarose gel, purified, and ligated into a linearized cloning vector. Hin [GenBank: see Availability and requirements section for URL] was cloned from Salmonella typhimurium Ames strain TA100 genomic DNA using forward primer 5'-GCATGAATTCGCGGCCGCTCTAGATGGCTACTATTGGGTATATTC and reverse primer 5'-ATGCCTGCAGGCGGCCGCAACTAGTTAATTCATTCGTTTTTTTATAC. HinLVA was generated by PCR amplification of Hin (BBa_J31000) using forward primer 5'-TCTGGAATTCGCGGCCGCATCTAGAGATG and reverse primer 5'-CTGCAGGCGGCCGCTACTAGTATTAAGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCATTCATTCGTTTTTTTATAC containing a ssrA LVA degradation tag (based on gfp(down, LVA) ).tetA(C) was cloned from vector pSB1AT3 using forward primer 5'-GCATTCTAGATGAAATCTAACAATGCGCTCATC and reverse primer 5'-ATGCACTAGTTAGGTCGAGGTGGCCCGGC; the amplicon was cloned using a XbaI/SpeI digest. Reversed parts were generated by PCR amplification of MIT Registry parts using a forward primer containing SpeI and a reverse primer containing XbaI: t