Engineered Optogenetic Fusion Proteins

Genes encoding for fusion proteins were assembled as a multicomponent cloning cassette from annealed oligonucleotides (IDT DNA) containing these elements (in order): BglII or NheI or BamHI — Secretion Signal/FLAG tag — HindIII — (αDTX or DTX-K or CONK1) — KpnI — Linker — LOV2-Jα(404–546) — NotI — PDGF-R-mCherry — XbaI or EcoRI. PDGF-R-mcherry was derived from pFU-MVIIA-PC (Addgene)^{10 (link)}. See Table 1 for sequence details. This cassette was inserted into the mammalian expression vector pcDNA3.1(+) (Invitrogen) using NheI / Xba restriction sites or a lentiviral vector containing the CAMKII promoter^{35 (link),36 (link)} using BamHI / EcoRI sites. The respective genes coding for rat Kv1.2 (Kv1.2), rat Kv1.1 (Kv1.1) or Shaker were amplified from Kv1.2-pBluescript, Shaker-pBluescript (gifts from Roderick Mackinnon), or BacMam Kv1.1 (Invitrogen) and inserted into pcDNA3.1(+) or pEGFP-N3 using BamHI / EcoRI or NheI/EcoRI, respectively. Both αDTX-lumitoxin and Kv1.2 cassettes were also inserted into the bidirectional expression vector pBI-CMV1 (Clontech) using BglII / XbaI and BamHI / NotI sites respectively, to drive expression from the same plasmid.