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Alice from Balloon Kid, while not Nintendo's first female main video game protagonist (being predated by Bubbles from Clu Clu Land and Samus from Metroid), was the first to be unambiguously female in all regions in which her game was released, as opposed to having her gender be a surprise reveal, or having an androgynous design and being assigned as female in a Western localization.
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Bubbles from Clu Clu Land is a she?
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https://www.thegameisafootarcade.com/wp-content/uploads/2017/02/Clu-Clu-Land-Game-Manual.pdf
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