Methods S2. Detailed pDOE vector schematics and cloning strategies. ( pg. 1 ) BiFC vector sets MCS1 MCS3 MCS2 p19/XT-mTq2 MASpro Pro::Ω NmVen210 OCSt CVen210 MASt NOSt X::tag—X::tag pDOE-01, -05, -09 X::tag—tag::X pDOE-02, -06, -10 tag::X—X::tag pDOE-03, -07, -11 tag::X—tag::X pDOE-04, -08, -12 Fluoroprotein fusion vector sets MCS1 MCS3 MCS2 p19 MASpro Pro::Ω mTurquoise2 OCSt mVenus MASt NOSt X::tag—X::tag pDOE-13, -17 X::tag—tag::X pDOE-14, -18 tag::X—X::tag pDOE-15, -19 tag::X—tag::X pDOE-16, -20

( pg. 2 ) Single sheet composite view of all four pDOE multiple cloning site arrangements This page provides the restriction enzyme recognition site sequences and their relationship to the open reading frame Restriction sites are directly above the enzyme name and the trailing underscore. (e.g. NcoI__ = ccATGG) MCS1 X-tag: M G R A L G T S G G S G G G S G . S R P G G . L ccATGGGGCGCGCCCTAGGAACTAGTGGAGGATCCGGAGGTGGGTCAGGA[NVen210]tctagacccgggggttaattaa NcoI__AscI____ SpeI__ BamHI_ XbaI__SmaI__ PacI____ AvrII_ BspEI_(dam dead) MCS1 tag-X: G A S G G G S G S M G R A L G T S . S R P G G . L [NVen210]GGAGCCAGTGGAGGTGGGTCAGGATCCATGGGGCGCGCCCTAGGAACTAGTtaatctagacccgggggttaattaa NcoI_ AscI____ SpeI_ XbaI__SmaI__ PacI____ BamHI_ AvrII_ MCS3 X-tag: M G S L R S H V T S G G S G G A . A I A I . caATGGGGTCCCTACGTAGTCACGTGACGTCCGGAGGTTCTGGTGGAGCT[SFS-CVen210]gcgatcgccatttaaat SanDI__SnaBI_ Eco721 BspEI_ AsiSI___ SwaI____ AatII_ MCS3 tag-X: S G A S G M G S L R S H V T S G . A I A I . I [CVen210-SFS]TCTGGAGCTTCAGGAATGGGGTCCCTACGTAGTCACGTGACGTCCGGAtaagcgatcgccatttaaat SanDI__SnaBI_ Eco721 BspEI_ AsiSI___ SwaI____ Deviation: In the “FRET” vectors the last Ala in the linker is Gly for X::mVenus Deviation: In the “FRET” vectors the mVenus::X constructs have a longer linker (SGAGSGGGSGM) and the stop codon is TGA

Comprehensive overview of the individual BiFC systems ( pg. 3 ) MCS1 (X::tag) M G R A L G T S G G S G G G S G . S R P G G . L ccATGGGGCGCGCCCTAGGAACTAGTGGAGGATCCGGAGGTGGGTCAGGA[NmVen210]tctagacccgggggttaattaa NcoI AscI AvrII SpeI BamHI BspEI (methyl blocked) XbaI SmaI PacI MCS3 (X::tag) M G S L R S H V T S G G S G G A . A I A I . caATGGGGTCCCTACGTAGTCACGTGACGTCCGGAGGTTCTGGTGGAGCT[SFS-CVen210]gcgatcgccatttaaat SanDI SnaBI PmlI AatII BspEI AsiSI SwaI MCS1 MCS3 MCS2 p19/XT-mTq2 MASpro Pro::Ω NmVen210 OCSt CVen210 MASt NOSt KpnI NruI XhoI Bsu36I *BstXI EcoRI 1) X::tag—X::tag pDOE-01, -05, -09 MCS1 (X::tag) M G R A L G T S G G S G G G S G . S R P G G . L ccATGGGGCGCGCCCTAGGAACTAGTGGAGGATCCGGAGGTGGGTCAGGA[NmVen210]tctagacccgggggttaattaa NcoI AscI AvrII SpeI BamHI BspEI (methyl blocked) XbaI SmaI PacI MCS3 (tag::X) S G A S G M G S L R S H V T S G . A I A I . I [CVen210-SFS]TCTGGAGCTTCAGGAATGGGGTCCCTACGTAGTCACGTGACGTCCGGAtaagcgatcgccatttaaat SanDI SnaBI PmlI AatII BspEI AsiSI SwaI MCS1 MCS2 MCS3 KpnI NruI p19/XT-mTq2 MASpro NmVen210 OCSt MASt CVen210 NOSt Pro::Ω XhoI Bsu36I *BstXI EcoRI 2) X::tag—tag::X pDOE-02, -06, -10

( pg. 4 ) 3) tag::X—X::tag pDOE-03, -07, -11 4) tag::X—tag::X MCS1 (tag::X) G A S G G G S G S M G R A L G T S . S R P G G . L [NVen210]GGAGCCAGTGGAGGTGGGTCAGGATCCATGGGGCGCGCCCTAGGAACTAGTtaatctagacccgggggttaattaa BamHI NcoI AscI AvrII SpeI XbaI SmaI PacI MCS3 (X::tag) M G S L R S H V T S G G S G G A . A I A I . caATGGGGTCCCTACGTAGTCACGTGACGTCCGGAGGTTCTGGTGGAGCT[SFS-CVen210]gcgatcgccatttaaat SanDI SnaBI PmlI AatII BspEI AsiSI SwaI MCS1 MCS2 XhoI MCS3 KpnI NruI p19/XT-mTq2 MASpro NmVen210 OCSt MASt CVen210 NOSt Pro::Ω Bsu36I *BstXI EcoRI 3) tag::X—X::tag pDOE-03, -07, -11 MCS1 (tag::X) G A S G G G S G S M G R A L G T S . S R P G G . L [NVen210]GGAGCCAGTGGAGGTGGGTCAGGATCCATGGGGCGCGCCCTAGGAACTAGTtaatctagacccgggggttaattaa BamHI NcoI AscI AvrII SpeI XbaI SmaI PacI MCS3 (tag::X) S G A S G M G S L R S H V T S G . A I A I . I [CVen210-SFS]TCTGGAGCTTCAGGAATGGGGTCCCTACGTAGTCACGTGACGTCCGGAtaagcgatcgccatttaaat SanDI SnaBI PmlI AatII BspEI AsiSI SwaI MCS1 MCS2 KpnI NruI Bsu36I MCS3 p19/XT-mTq2 MASpro NmVen210 OCSt CVen210 MASt NOSt Pro::Ω XhoI *BstXI EcoRI 4) tag::X—tag::X pDOE-04, -08, -12

Cloning options for MCS1 BamHI = ..ATCC (Ser) BclI = ..ATCA (Ser) ( pg. 5 ) Cloning options for MCS1 BamHI = ..ATCC (Ser) BclI = ..ATCA (Ser) BglII = ..ATCT (Ser) MCS1 (X::tag) M G R A L G T S G G S G G G S G . S R P G G . L ccATGGGGCGCGCCCTAGGAACTAGTGGAGGATCCGGAGGTGGGTCAGGA[NmVen210]tctagacccgggggttaattaa NcoI AscI AvrII SpeI BamHI BspEI (methyl blocked) XbaI SmaI PacI Insert PCR product cut with: NcoI = ..ATGG BspHI = ..ATGA PciI = ..ATGT If your ORF is ..ATGC, insert a novel 2nd codon starting with G,A, or T such as Gly (GGN), e.g. ATG GGN [CNN] Tag removal and other conjugations via compatible ends: AvrII SpeI XbaI in frame NEB BspEI will not cut. ThermoFisher BspEI will cut this site and the site in MCS3; religating creates a MCS1/3 hybrid for tag exchange, single expression, etc. Additional options: 1) Erase the NcoI site in the ORF by overlap PCR. This is a better option overall, and allows for easy retrieval of the ORF. 2) Utilize the rare cutting AscI site.

Cloning options for MCS3 AatII out-of-frame cohesive cut ( pg.6 ) Cloning options for MCS3 AatII out-of-frame cohesive cut ZraI out-of-frame blunt cut MCS3 (X::tag) M G S L R S H V T S G G S G G A . A I A I . caATGGGGTCCCTACGTAGTCACGTGACGTCCGGAGGTTCTGGTGGAGCT[SFS-CVen210]gcgatcgccatttaaat SanDI SnaBI PmlI AatII BspEI AsiSI SwaI PCR product contribution underlined: SanDI = GGGTCCC RsrII = CGGTCCG AvaII = GGTCCn PpuMI = RGGTCCY PmlI/Eco72I in-frame blunt cut BspEI compatible sites: AgeI/BshTI = ACCGGT XmaI = CCCGGG NgoMIV = GCCGGC (plus others*) *BsaWI (WCCGGW) BsrFI (RCCGGY) If the ORF for your gene is M[X] where x = L, R, P, H, Q your primer is ATG GGG TCC [X] = MGS[X] with a SanDI site If the ORF for your gene is M[X] where x = G, A, V, E, D your primer is ATG CGG TCC [X] = MRS[X] with an RsrII site RG after ligation If the second residue of your ORF is not one of the ten residues noted above, choose one to add as a third inserted codon, e.g. MGSG[X]… * For SanDI, your primer will be ATG GGG TCC CNN [native ORF 2nd residue] * For RsrII, your primer will be ATG CGG TCC GNN [native ORF 2nd residue]

Constructions are made in the same vector, ( pg. 7 ) Constructions are made in the same vector, and the pDOE system can be thought of as one large MCS. For example: 1) Cloning into NcoI + an MCS3 upstream site yields a single cassette ORF::CVen210 construct (e.g. NcoI + MCS3 BspEI) — Orientations besides the one shown will give other products 2) Cloning into NcoI + AsiSI yields a single cassette untagged protein 3) Ligating the blunt cut sites of SmaI + SwaI deletes the third cassette (and tails MCS1 with the NOSt) MCS1 (X::tag) M G R A L G T S G G S G G G S G . S R P G G . L ccATGGGGCGCGCCCTAGGAACTAGTGGAGGATCCGGAGGTGGGTCAGGA[NmVen210]tctagacccgggggttaattaa NcoI AscI AvrII SpeI BamHI BspEI (methyl blocked) XbaI SmaI PacI BspEI MCS1/3 merge also allows fluoroprotein exhange: Cloned MCS1 ORF::mTq2  cut/religate = ORF::mVenus This merge can also doubly fluorotag a protein: Cloned MCS1 mTq2 ORF  cut/religate = mTq2::ORF::mVenus MCS3 (X::tag) M G S L R S H V T S G G S G G A . A I A I . caATGGGGTCCCTACGTAGTCACGTGACGTCCGGAGGTTCTGGTGGAGCT[SFS-CVen210]gcgatcgccatttaaat SanDI SnaBI PmlI AatII BspEI AsiSI SwaI