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Construction of an expression library comprising constitutively active mutant small GTPases. Cloning scheme for the constitutively active mutant small GTPase library. Each GTPase was amplified into two fragments by using mutagenic primers in the GTPase domain. The PCR products were then fused to the linearized backbone vector with an HA-tag on either the N- or C-terminal side of the insert site simultaneously through 3-fragment Gibson assembly reaction.
