I guess, you have done the initial cloning in a strain for DNA amplification, e.g. XL1blue. This plasmid should be still available. A digestion will proof that you transformed a vector with insert into BL21.

My suggestion: Repeat your transformation in BL21, grow your bacteria and check for the presence of your insert. If it is present you made a mistake with your first attempt. If it is gone, your construct is very unstable and you may optimize your protocol, e.g. grow bacteria at lower temperatures.

I presume you confirmed that the initial clone you made a glycerol stock carried a plasmid with an insert? It could also be that your initial clone was actually a mixture of plasmids with insert and plasmids without an insert and it sergregated out to the plasmid without insert.

I recommended Dr. Uwe Borgmeyer's description. Sometimes bacteria remove a part of the cloned gene when keeping the gene on the plasmid is so stressful. Even if the plasmid was OK before making glycerol stock, the plasmid may change during culture to make glycerol stock or for the next experiment. Low copy plasmid may solve the problem.

I agree with Dr. Benedik in that you may have transformed a mix of plasmids with and without the gene of interest. I believe it may be more difficult for the bacteria to remove an entire gene than just add a few random mutations to inactivate it.