The PCI-324 drivers are multi-client. Therefore, more than one application may use the PCI-324 card at one time as long as they are using unique ports. One application such as Unity or Retro can use the PCI-324 ASIO Driver while another application such as Cakewalk can be using the PCI-324 Wave driver at the same time. However, these applications can not share the ports. If one application is using ports 1-2, the other application must use another port such as 3-4.
In most cases you will get an explicit warning which explains this as soon as you try to share a port with two applications. In the case of sharing with a software synth you may not see this warning as you can use a software synth without having the application open in the background. A symptom you may see in this situation is losing audio output from one application when you assign a track to your software synth. To resolve the problem, configure your software synth to use outputs 3-4 while your other application uses outputs 1-2.