Yes. If you are talking about an axial constraint for the cylinder eye and the cylinder pull tab ... the centers are about .025" out of alignment, so you will get an error. You can see it if you look thru the holes and zoom in.
A couple of things. You are missing many constraints. Constraints are very important in modelling. Not just one piece to another, but in the respective workspace. After un-anchoring the outside gate tubes the entire assembly can move about the workspace.
Secondly, the air cylinder assembly itself (when in its own workspace) is not fully constrained and free to move and rotate about. I would start there. Also, the hole in the cylinder eye is not fully constrained. I've not used the "limited" motion constraint feature that you used on the cylinder, so I am not sure if the "limits" translate from sub-assembly to major assembly levels. I'm sure others can address that if need be.
I would suggest using the workspace geometry (planes and axes) to your advantage. For example, construct your cylinder about an axis, much like you did the rod and maybe use another axis for the fixed end hole center. Same with the eye. Use an axis for the hole center and extrude about a mid plane. This type of approach makes the assembly and constraint part much easier.
Hope that helps.