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Painted 1489-90, the painting was eventually purchased by Ezio Auditore da Firenze and placed in the gallery at the Villa Auditore. The painting was later lost during the Papal attack on Monteriggioni, led by the Templar Cesare Borgia.
Six years after the attack on Monteriggioni, in 1506, Ezio Auditore – whilst looking for a kidnapped Leonardo – learned that the Lady with an Ermine held part of a map identifying the location of the Temple of Pythagoras, and that the painting was in the possession of a Florentine merchant based in Rome. Upon approaching the merchant, Ezio was shocked to discover it was his sister Claudia's former fiancé – Duccio de Luca.
Despite Ezio's attempts to remain civil, Duccio's insults regarding Claudia saw him receive a stout punch to the face. After a prolonged fistfight between the Assassin and Duccio's entourage, Ezio retrieved the painting, as Duccio lay on the ground unconscious.