The build and design rules for BoP.

•Only Bird-of-Prey-Compliant (BoP-ized) genes can be assembled using Bird of Prey.
•If a gene is not in our library we can convert nearly any gene of interest into Bird of Prey using our vector building service.
•No Duplicate Transcription Units
•No Duplicate IRES’s in a Transcription Unit

•Expression efficiency decreases with a larger number of ORFs in a single Transcription Unit.
•Limit each Transcription Unit to three ORFs (and two IRESes) maximum to prevent poor expression levels.
•Lower expression levels occur at the 3' end of IRES Shuttle Vectors due to ribosomal mechanics in translation – use this to your advantage.

•IRES Shuttle Vectors contain two Effector positions: 5' and 3' of the IRES.
•When building multi-effector constructs with IRES: First subclone the Effector ORF into the 3' position,- Then subclone the second ORF into the 5' position

•All enzymes needed for Bird of Prey can be found on NEB our enzymes tab.
•Follow our protocol suggestions for enzyme quantities. This will prevent inefficient cutting.

•Keep aliquots of each of your construction steps (including the original module) to use for making multiple combinations using the same modules.
•Validate each step of your construction to benchmark as you go.
•If your insert is around the same size as the vector backbone, you can use the AseI enzyme to linearize and identify the gel band more easily during restriction digests.

•Test Controller Vectors in target cells before building multi-effector/multigene constructs.
•Experiment with different configurations and order of inserts for optimal expression.
•Consider using different promoters and regulatory elements to achieve desired expression levels.
•When targeting multiple cell types in vivo, use different cell-type controllers with multiple effectors.

•Bird of Prey is designed to mimic natural gene architecture and function as closely as possible.
•When considering regulatory elements, note that our R domains consist of DNA-encoded elements that control 1) Pre-Transcription and 2) Transcription; and to the RNA-encoded elements that control 3) Post-transcription and 4) Pre-Translation, and to the Amino Acid-encoded elements that control 5) Translation and 6) Post-translational biological activities.
•Our R (Transcription Regulator) Domain is defined by DNA regulatory elements at the 5’ end of the gene and by RNA elements at the 3’ end. This order is reversed in the P (Transcription Processing) Domain.