MySQL 8.0

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CREATE TABLE `locations`(
    `location_id` INT NOT NULL AUTO_INCREMENT,
    PRIMARY KEY (`location_id`)
);
CREATE TABLE IF NOT EXISTS `routes`(
    `route_id` INT NOT NULL AUTO_INCREMENT,
    `location_id_1` INT NOT NULL,
    `location_id_2` INT NOT NULL,
    FOREIGN KEY (`location_id_1`) 
        REFERENCES `locations` (`location_id`)
        ON UPDATE CASCADE
        ON DELETE CASCADE,
    FOREIGN KEY (`location_id_2`) 
        REFERENCES `locations` (`location_id`)
        ON UPDATE CASCADE
        ON DELETE CASCADE,
    UNIQUE KEY( (LEAST(`location_id_1`, `location_id_2`)),
                (GREATEST(`location_id_1`, `location_id_2`))),
    PRIMARY KEY (`route_id`)
);
INSERT INTO locations VALUES (1), (2);
INSERT INTO routes VALUES (1,1,2);

Records: 2  Duplicates: 0  Warnings: 0


INSERT INTO routes VALUES (2,2,1);

Duplicate entry '1-2' for key 'routes.functional_index'