Put it immediately after the invitation or offer, so the refusal answers the setup without a gap. For a double rejection, follow it with Nope, and for a comic reversal, open with Okay Let's Go before cutting to this clip as the real answer.
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This is a brief spoken clip, just under two seconds long, in which a voice delivers the line “No, not interested” in a flat, unenthusiastic tone. It lands as a blunt, slightly awkward brush-off, funny because it offers no excuse or softening at all. The deadpan delivery makes the refusal feel final, like a door closing on the conversation before it can continue.