We use a proprietary homology-based cloning method to assemble our clonal genes, so restriction sites are not critical to cloning and you do not need to consider Twist's cloning design when designing your insert. However, if your insertion site is between two restriction sites, include these as part of the vector backbone, to ensure features are in frame.

There are two options for using restriction enzyme cloning.

Option 1 - Include Restriction Sites on the insert

Include the whole restriction enzyme site on either end of the insert sequence. The restriction enzyme sites will be part of the inserts in silico.

When you order genes, you must include restriction enzyme sites on the inserts.  In this custom vector onboarding scenario, if you exclude restriction enzyme sites on the genes in your order, the restriction enzyme sites will be completely absent from your final product.

Option 2 - Include Restriction Sites on the vector

Do not include restriction enzyme sites on the insert sequence. Include each whole restriction enzyme site on the Vector + Insert box in Custom Vector onboarding form. The restriction enzyme sites will be part of the vector in silico.

When you order genes, do not include restriction enzyme sites on the inserts.  In this custom vector onboarding scenario, if you include restriction enzyme sites on the genes in your order, the restriction enzyme sites will be duplicated in your final product.