Fig. 2

ImiRP input user interface. a The user is first directed to create a project and specify project parameters. The project can be named, and in this example we have named the project “ImiRP Test”. A DNA or RNA sequence must be input along with information about the species of interest. The input sequence will be displayed for viewing. Finally, at least one “mutation site”, the region into which mutations will be introduced, is specified by typing the sequence position complementary to miRNA position 7 into the textbox. All selected mutation sites, complementary to miRNA positions 2-7, appear bolded in the displayed original input sequence for inspection. In this example, a segment of the mouse Pax6 3’UTR and three hypothetical predicted target sites have been used as input. b Upon creation of a project, the user is asked to specify mutation parameters. In the “Mutation Strategy” tab, nucleotides to use for mutation and number of nucleotide changes per specified mutation site can be selected. In the “Define Invalid Sites” tab, users can specify the types of newly created miRNA target sites they do not want present in their final mutant sequence. In this case, any mutant sequences provided as output will contain two adjacent nucleotide changes per specified target site using all four nucleotides for mutation, and no mutant sequences containing newly created 8mer, 7mer-m8, or 7mer-A1 predicted target sites will be provided as output