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Artificial chromosomes were previously generated for use in bacteria, protists, yeast and human cells. A Tetrahymena thermophila artificial chromosome could serve as a versatile platform to study diverse aspects of Tetrahymena biology and beyond. Here, we placed a C3-type rDNA replication origin and telomere sequences from T. thermophila into a pNeo4 vector, producing the first T. thermophila macronuclear artificial chromosome (TtAC1). Circular or linear forms of TtAC1 can be stably transformed into both vegetative and conjugative T. the