The anthocyanidin synthase DNA sequence from Fragaria x ananassa was the origin DNA sequence for the construction of your non-engineered ANS. To improve the expression efficiency of the Fragaria x ananassa ANS in Escherichia coli we optimised the DNA sequence towards the codon usage of E. coli. After addition of Prefix, RBS B0034 and Suffix to the optimised ANS DNA sequence the final sequence was ordered at IDT gensynthesis service. When the DNA was arrived we performed a PCR with the Primers X and X to amplify your construct. Then we digested the construct with EcoRI and PstI and ligated it with T4 Ligase into pSB1A2 which was restricted before with EcoRI and PstI. After Ligation we transformed the pSB1A2-RBS-ANS construct via heat shock transformation. For spreading out we used AMP-LB agar plates. After incubating overnight at 37°C we analysed obtained colonies by colony-PCR and agarose gel electrophoresis. We inoculated positive colonies in 5 ml LB with AMP. After incubating for 16 h at 37°C we prepped plasmids. For further verification we send the samples to Eurofins for sequencing.