The　complexity　of　genetic　circuits　has　not　seen　a　significant　increase　over　the　last　decades,　even　with　the　rapid　development　of　synthetic　biology　tools.　One　of　the　bottlenecks　is　the　limited　number　of　orthogonal　transcription　factor–operator　pairs.　Researchers　have　tried　to　use　aptamer–ligand　pairs　as　genetic　parts　to　regulate　transcription.　However,　most　aptamers　selected　using　traditional　methods　cannot　be　directly　applied　in　gene　circuits　for　transcriptional　regulation.　To　that　end,　we　report　a　new　method　called　CIVT-SELEX　to　select　DNA　aptamers　that　can　not　only　bind　to　macromolecule　ligands　but　also　undergo　significant　conformational　changes,　thus　affecting　transcription.　The　single-stranded　DNA　library　with　affinity　to　our　example　ligand　human　thrombin　protein　is　first　selected　and　enriched.　Then,　these　ssDNAs　are　inserted　into　a　genetic　circuit　and　tested　in　the　in　vitro　transcription　screening　to　obtain　the　ones　with　significant　inhibitory　effects　on　downstream　gene　transcription　when　thrombins　are　present.　These　aptamer–thrombin　pairs　can　inhibit　the　transcription　of　downstream　genes,　demonstrating　the　feasibility　and　robustness　of　their　use　as　genetic　parts　in　both　linear　DNAs　and　plasmids.　We　believe　that　this　method　can　be　applied　to　select　aptamers　of　any　target　ligands　and　vastly　expand　the　genetic　part　library　for　transcriptional　regulation.　©　2023　by　the　authors.