Have you tried your while loop like this?

my $locus;

while (<$in>) {
    if ( /LOCUS\s+(\w+)/ ) {
        $locus = $1;
    }
    elsif ( /\^\^/ ) {
        if ( $locus ) {
            s/\^\^/>$locus\n^^\n/g;
        }
        else {
            warn "Found ^^ without a preceding LOCUS value\n";
        }
    }
    print $out $_;
}
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Part of the problem is that you only want to assign to $locus when you have just read that particular sort of line from the file. If you do this sort of assignment on every line:

        ( $locus ) = ( /LOCUS\s+(\w+)/ );
[download]

then $locus will get cleared every time your regex fails to match a given line of input. So see if the match will work first, and if it does, then assign the result to $locus.

Another part is that you'll want to know (via STDERR) if you get a situation where a locus value should be plugged in but you don't have one yet.

(I hope I understood the question -- not sure that I did...)