Assuming everything is in the right ballpark then these three have the greatest overall affect on performance:
1) Tyre Pressures 2) Carb Settings 3) Front-end geometry
If we're just talking getting the kart balanced from a good baseline:
1) Tyre Pressures 2) Front-end geometry 3) Rear width
If you're starting from scratch then:
1) Seat Position 2) Front-end geometry 3) Carb 4) Pressures 5) Rear width 6) Gearing
Gearing is always the last of my concerns as everything else ultimately dictates the gearing you need to run and you'll be changing it throughout the day anyway. However, you must remember that gearing can have a fairly significant effect on the balance of the kart coming off the corners.
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