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The COSPLAY primers design software has been developed under MatLab 2017b to simplify and automate the addition of new personalized modules to the COSPLAY modules collection. The software can be downloaded at:______________(MatLab or standalone files). The standalone version is to use when no MatLab is available on the running machine. (this version is longer to download because it also instal the MatLab 2017 running environment).

The main 5 functions of the COSPLAY software are:

1. Automatically designing the most optimal homologous primers that are intended to initially amplify the module sequence. Many criteria (Primer Tm, length, self complementarity, 3' complementarity, matrix complementarity, 3' matrix complementarity, GC %, GC clamps, 3' stability, 3' GC%, primers pair Tm difference, cross complementarity, cross 3' complementarity) are used for this optimisation. The basic rules of optimisation are similar to those of the Primer3 software (Rozen and Skaletsky, 2000).
2. Adding specific, module type dependant tails to the homologous primers. There are 6 different types of modules each requiring 2 unique, specific tails for the corresponding forward and reverse primers. These tails contain each: i) PUC57 homologous sequence (module type independant) used to integrate the module into a carrying plasmid and ii) a BsaI site and associated linker sequence (module type specific) used to assemble the modules in a particular order.
3. Detecting BsaI sites in the module sequence and replacing with silent substitutions whenever it is possible (or indicating alternative way to discard the sites).
4. Estimating the best PCR temperature conditions. The Tm of the primers is calculated using an accurate thermodynamic approach (SantaLucia, 1998). This Tm is optimised for Q5 polymerase which is recommended to use in our protocol (if standard Taq polymerase is used the Tm have to be lowered by 7°C). The effect of the buffer salt concentration is also calculated (Owczarzy et al., 2004). In case there are some nucleotides substitutions the mismatch effect on the Tm is also precisely estimated (Allawi and SantaLucia, 1997, 1998a, 1998b, 1998c, 1998d; Peyret et al., 1999).
5. Displaying a full report about the primers pair quality when the entire tailed primers are automatically designed or indicating potential quality problems when only tails are added to already existing primers.