SUPPLEMENTARY MATERIAL

Transcription

1 Activity [RLU] Activity [RLU] RAB:TALA TALA:RAB TALB:RAB RAB:TALB RAB:TALB TALB:RAB TALA:RAB RAB:TALA Activity [nrlu] Activity [nrlu] SUPPLEMENTARY MATERIAL a b ng ng c [a]-cmv_fluc [25 ng] e [b]-cmv_fluc [25 ng] d Control Pristinamycin [2 mg/l] Control Erythromycin [2 mg/l] f PIP:RAB [ng] [pir]-p CMV_TALB:RAB [ng] [cb]-p CMV_fLuc [20 ng] E:RAB [ng] [etr]-p CMV_TALA:RAB [ng] [ac]-p CMV_fLuc [20 ng] Supplementary Figure 1 Designed TALE repressors strongly inhibit reporter gene expression. (a,b) TALE-A (a) and TALE-B (b) repressor efficiency and mutual orthogonality were tested using luminometry. N- and C-terminal RAB fusion variants of TALE repressors were tested. Increasing amounts of TALE repressors were co-transfected into HE293T cells along with the firefly luciferase reporter plasmid containing ten copies of appropriate binding sites upstream of the CMV promoter. (c,e) TALE repressors were coupled with pristinamycin- (c) and erythromycin-inducible (e) transcriptional repressors (PIP:RAB and E:RAB, respectively) as indicated in the schematic representation. (d,f) Response to chemical inducers was evaluated using luminometry. The firefly luciferase reporter plasmid with ten copies of appropriate binding sites upstream of the CMV promoter was co-transfected into HE293T cells along with increasing amounts of plasmids encoding TALE repressor constructs and plasmids, encoding the corresponding inducible transcriptional repressor in a 2:3 ratio. Values represent the mean and standard deviation of three replicates.

2 Activity [RLU] Activity [RLU] Activity [RLU] Activity [RLU] a b c [ng] TALA:VP16 TALB :VP16 [a]-min_fluc [25 ng] [ng] TALB:VP16 TALB :VP16 [b]-min_fluc [25 ng] e d f Control Pristinamycin [2 mg/l] Control Erythromycin [2 mg/l] PIP:RAB [ng] [pir]-p CMV_TALA:VP16 [ng] [ac]-p CMV_fLuc [20 ng] PIP:RAB [ng] [pir]-p CMV_TALA:VP16 [ng] [ac]-p min_fluc [20 ng] E:RAB [ng] [etr]-p CMV_TALB:VP16 [ng] [cb]-p CMV_fLuc [20 ng] E:RAB [ng] [etr]-p CMV_TALB:VP16 [ng] [cb]-p min_fluc [20 ng] Supplementary Figure 2 Designed TALE activators display strong activation of a minimal promoter. (a,b) TALE-A (a) and TALE-B (b) activator efficiency and mutual orthogonality were tested using luminometry. Increasing amounts of TALE activators were co-transfected into HE293T cells along with 25 ng of the firefly luciferase reporter plasmid with ten copies of appropriate binding sites upstream of a minimal promoter. (c,e) TALE activators were coupled with pristinamycin- (c) and erythromycin-inducible (e) transcriptional repressors (PIP:RAB and E:RAB, respectively) as indicated in the schematic representation. (d,f) Response to chemical inducers was evaluated using luminometry. Twenty nanograms of a firefly luciferase reporter plasmid containing ten copies of appropriate binding sites upstream of the CMV (left panels) or a minimal promoter (right panels) were co-transfected into HE293T cells along with increasing amounts of plasmids encoding TALE activator constructs and plasmids encoding the corresponding inducible transcriptional repressor in a 2:3 ratio. Values represent the mean and standard deviation of three replicates.

3 Concentration Concentration Concentration Concentration a b c d ER - ER cooperativity 1; leakage 0.00 PI - PI - PI ER - - ER - PI ER Time [hours] cooperativity 1; leakage 0.01 (Pmin); 0.1 (PCMV) PI ER - PI Supplementary Figure 3 Deterministic modeling demonstrates that positive feedback and competitive binding are the key features for achieving bistability with non-cooperative DNA binding elements. Ordinary differential equation (ODE)-based models of the competitive feedback switch with the repression leakage parameter varying from 0% (left panels) to 1% (right panels). To account for the non-cooperative TALE binding, Hill coefficients n i = 1 are assumed. (a,b) Simulations of chemical inducer pulse (PI, ER) and growth in the absence of an inducer (-) demonstrate robustness to incomplete repression in contrast to the mutual repressor switch models (Fig. 1c). Bistability is achieved despite low Hill coefficients and incomplete repression. (c,d) Switching between the two states (PI, ER). Details on the model are available in Supplementary Note 1.

4 mcitrine [FI] mcitrine [FI] mcitrine [FI] mcitrine [FI] a b c mcitrine TagBFP TagBFP [FI] d mcitrine TagBFP TagBFP [FI] mcitrine TagBFP TagBFP [FI] mcitrine TagBFP TagBFP [FI] Supplementary Figure 4 Control experiments for the competitive feedback switch show that all components of the genetic circuit function as expected. (a,b) Excess TALE repressors and activators corresponding to one of the two states induce only that selected state, as depicted in the schematic presentations (upper panels), experimentally determined with confocal microscopy and flow cytometry (lower panels). (c,d) Addition of only one of the chemically inducible repressors (E:RAB or PIP:RAB) in the absence of inducers represses only the selected state, thus inducing the opposite state, as depicted in the schematic presentations (upper panels), confirmed with confocal microscopy and flow cytometry (lower panels). Images and histograms are representative of at least three independent experiments. Scale bar = 50 µm.

5 mcitrine [FI] Cell number [%] mcitrine [FI] Cell number [%] mcitrine mcitrine TagBFP TagBFP a PI added ER added b ER PI 3 days 6 days 9 days c PI ER 3 days 6 days 9 days d Both TagBFP mcitrine e Both TagBFP mcitrine TagBFP [FI] days ER PI TagBFP [FI] days PI ER Supplementary Figure 5 Confocal microscopy and flow cytometry analyses of controlled switching between stable states. (a) A schematic presentation of switching between stable states. (b,c,d,e) Addition of an inducer molecule immediately after transfection causes expression of the selected state. Replacement of the initial inducer molecule with the opposite chemical inducer three days after transfection caused a slow, but clear, transition of the initial state into the opposite state, confirmed with confocal microscopy and flow cytometry (b,d) Erythromycin to pristinamycin; (c,e) pristinamycin to erythromycin. Scale bar = 50 µm. The microscopy and flow cytometry results are representative of at least three independent experiments. The number of cells expressing specific reporters (as obtained from flow cytometry) is presented in the histograms.

6 a b Supplementary Figure 6 The competitive feedback switch based on the minimal promoters toggles faster than the switch based on the constitutive CMV promoters. Simulations of toggling from the (a) pristinamycin- to erythromycin-induced state (PI to ER) and (b) from the erythromycin- to pristinamycininduced state (ER to PI) with the stochastic model. The competitive feedback switch with minimal promoters reaches a stable steady state in significantly shorter time after the introduction of the second inducer. Details of the model are available in Supplementary Note 2.

7 mcitrine mcitrine [FI] mcitrine [FI] Cell number [%] TagBFP mcitrine mcitrine [FI] mcitrine [FI] TagBFP Cell number [%] a PI added ER added b PI ER 3 days 6 days 10 days 13 days c PI (3 d) ER (15 d) 100 PI ER TagBFP [FI] TagBFP [FI] Time [d] d ER PI 3 days 6 days 10 days 13 days e Both TagBFP mcitrine ER (3 d) PI (15 d) 100 ER PI TagBFP [FI] TagBFP [FI] Time [d] Supplementary Figure 7 Toggling between the two stable states by the competitive feedback switch based on minimal promoters. (a) A schematic presentation of switching between stable states. (b,c,d,e) Addition of the inducer molecule promotes expression of the selected state. Replacement of the initial inducer molecule (PI or ER) with the opposite chemical inducer (ER or PI) three days after transfection triggered a transition from the initial state to the opposite state, which was confirmed with confocal microscopy and flow cytometry. (b,c) Switch from pristinamycin to erythromycin (PI to ER); (d,e) switch from erythromycin to pristinamycin (ER to PI). Scale bar = 50 µm. The microscopy and flow cytometry results are representative of at least three independent experiments. The number of cells expressing specific reporters obtained from flow cytometry is presented in the histograms (c and e, right panel).

8 Supplementary Table 1 Plasmids used in the study. Unless stated otherwise, all constructs containing the CMV promoter were inserted in the pcdna3 backbone, and all constructs containing the minimal promoter were inserted in the pgl4.16 backbone. No. Plasmid name and construct illustration Plasmid description [CMV]_l2_TALA:RAB [CMV]_l2_TALB:RAB [CMV]_l2_RAB: TALA [CMV]_l2_RAB: TALB [CMV]_l2_TALA:VP16 [CMV]_l2_TALB:VP16 Plasmid ensures constitutive expression of TAL effector A with 2xNLS signal and RAB repression domain on the C-terminal end (TALA:RAB). Plasmid ensures constitutive expression of TAL effector B with 2xNLS signal and RAB repression domain on the C-terminal end (TALB:RAB). Plasmid ensures constitutive expression of TAL effector A with 2xNLS signal and RAB repression domain on the N-terminal end (RAB:TALA). Plasmid ensures constitutive expression of TAL effector B with 2xNLS signal and RAB repression domain on the N-terminal end (RAB:TALB). Plasmid ensures constitutive expression of TAL effector A with 2xNLS signal and VP16 activation domain (TALA:VP16). Plasmid ensures constitutive expression of TAL effector B with 2xNLS signal and VP16 activation domain (TALB:VP16) x[a]_l4_[CMV]_l2_fLuc 10x[b]_l4_[CMV]_l2_fLuc Plasmid ensures expression of reporter (firefly luciferase) in the absence of TALA:RAB or RAB:TALA. 10 repeats of TALA binding sites [a] are cloned upstream and the reporter gene downstream of a CMV promoter. Plasmid ensures expression of reporter (firefly luciferase) in the absence of TALB:RAB or RAB:TALB. 10 repeats of TALB binding sites [b] are cloned upstream and the reporter gene downstream of a CMV promoter. 10x[a]_l3_[min]_l1_fLuc Plasmid ensures expression of reporter (firefly luciferase) in the presence of TALA:VP repeats of TALA binding sites [a] are cloned upstream and the reporter gene downstream of a minimal promoter. 10x[b]_l3_[min]_l1_fLuc Plasmid ensures expression of reporter (firefly luciferase) in the presence of TALB:VP repeats of TALB binding sites [b] are cloned upstream and the reporter gene downstream of a minimal promoter. 3x[pir]_l4_[CMV]_l2_TALB:RAB 3x[pir]_l4_[CMV]_l2_TALA:VP16 8x[etr]_l4_[CMV]_l2_TALA:RAB 8x[etr]_l4_[CMV]_l2_TALB:VP16 [CMV]_l2_PIP:RAB Plasmid ensures expression of TALB:RAB in the absence of PIP:RAB or in the presence of pristinamycin. 3 repeats of the PIP binding site [pir] are cloned upstream and TALB:RAB downstream of a CMV promoter. Plasmid ensures expression of TALA:VP16 in the absence of PIP:RAB or in the presence of pristinamycin. 3 repeats of the PIP binding site [pir] are cloned upstream and TALA:VP16 downstream of a CMV promoter. Plasmid ensures expression of TALA:RAB in the absence of E:RAB or in the presence of erythromycin. 8 repeats of the E binding site [etr] are cloned upstream and TALA:RAB downstream of a CMV promoter. Plasmid ensures expression of TALB:VP16 in the absence of E:RAB or in the presence of erythromycin. 8 repeats of the E binding site [etr] are cloned upstream and TALB:VP16 downstream of a CMV promoter. Plasmid ensures expression of PIP (pristinamycin inducible protein) with 3xNLS signal and RAB repression domain (PIP:RAB). 16 [CMV]_l2_E:RAB Plasmid ensures expression of E (erithromycin inducible protein) with3x NLS signal and RAB repression domain (E:RAB) x[b]_l4_[CMV]_l2_TALA:RAB:t2A:mCit Plasmid ensures expression of TALA:RAB and mcitrine in the absence of TALB:RAB. 10 repeats of TALB binding sites [b] are cloned upstream and TALA:RAB linked to mcitrine reporter via the t2a sequence downstream of a CMV promoter. 10x[a]_l4_[CMV]_l2_TALB:RAB:t2A:BFP Plasmid ensures expression of TALB:RAB and TagBFP in the absence of TALA:RAB. 10 repeats of TALA binding sites [a] are cloned upstream and TALB:RAB linked to TagBFP reporter via the t2a sequence downstream of a CMV promoter. 10x[a]_l4_[CMV]_l2_TALA:VP16 Plasmid ensures expression of TALA:VP16 in the absence of TALA:RAB. 10 repeats of TALA binding sites [a] are cloned upstream and TALA:VP16 downstream of a CMV promoter x[b]_l4_[CMV]_l2_TALB:VP16 Plasmid ensures expression of TALB:VP16 in the absence of TALB:RAB. 10 repeats of TALB binding sites [b] are cloned upstream and TALB:VP16 downstream of a CMV promoter x[b]_l3_[min]_l1_TALA:RAB:t2A:mCit Plasmid ensures expression of TALA:RAB and mcitrine in the presence of TALB:VP16. 10

9 repeats of TALB binding sites [b] are cloned upstream and TALA:RAB linked to mcitrine via the t2a sequence downstream of a minimal promoter. 10x[a]_l3_[min]_l1_TALB:RAB:t2A:BFP Plasmid ensures expression of TALB:RAB and TagBFP in the presence of TALA:VP repeats of TALA binding sites [a] are cloned upstream and TALB:RAB linked to TagBFP via the t2a sequence downstream of a minimal promoter. 10x[b]_l3_[min]_l1_TALA:RAB:t2A:fLuc 10x[a]_l3_[min]_l1_TALB:RAB:t2A:fLuc Plasmid ensures expression of TALA:RAB and firefly luciferase in the presence of TALB:VP repeats of TALB binding sites [b] are cloned upstream and TALA:RAB linked to firefly luciferase via the t2a sequence downstream of a minimal promoter. Plasmid ensures expression of TALB:RAB and firefly luciferase in the presence of TALA:VP repeats of TALA binding sites [a] are cloned upstream and TALB:RAB linked to firefly luciferase via the t2a sequence downstream of a minimal promoter x[b]_l3_[min]_l1_TALA:RAB 10x[a]_l3_[min]_l1_TALB:RAB 10x[a]_l3_[min]_l1_TALA:VP16 10x[b]_l3_[min]_l1_TALB:VP16 [CMV]_mCherry [CMV]_iRFP670 [T]_rLuc Plasmid ensures expression of TALA:RAB in the presence of TALB:VP repeats of TALB binding sites [b] are cloned upstream and TALA:RAB downstream of a minimal promoter. Plasmid ensures expression of TALB:RAB in the presence of TALA:VP repeats of TALA binding sites [a] are cloned upstream and TALB:RAB downstream of a minimal promoter. Plasmid ensures expression of TALA:VP16 in the presence of TALA:VP repeats of TALA binding sites [a] are cloned upstream and TALA:VP16 downstream of a minimal promoter. Plasmid ensures expression of TALB:VP16 in the presence of TALB:VP repeats of TALB binding sites [b] are cloned upstream and TALB:VP16 downstream of a minimal promoter. pmcherry-c1 (Clontech) with mcherry gene insert. Plasmid ensures constitutive expression of mcherry, and was used to normalize levels of transfection in confocal microscopy experiments. pcdna3with irfp670 gene insert. Plasmid ensures constitutive expression of irfp, and was used to normalize levels of transfection in flow cytometry experiments. phrl-t (Promega) with Renilla luciferase gene insert. Plasmid ensures constitutive expression of Renilla luciferase, and was used to normalize levels of transfection in luciferase assay experiments.

10 Supplementary Table 2 Nucleotide and amino acid sequences of the constructs used in the study. Nucleotide/ amino acid sequence P CMV CMV promoter from plasmid vector pcdna3 acattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctg gctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacgg taaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgac cttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgact cacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacg caaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctc P min Minimal promoter tagagggtatataatggaagctcgacttccag 10x[a] TALA binding site [a] tttactgctgctcccgct tttactgctgctcccgcttaaagagcagaaactcatgctggcagccggggtaccgcaggcaagtcgctttactgctgctcccgcttaaagagcagaaactcaatgga atcacggcggccgcccgtatctcatcctttactgctgctcccgcttaaagagcagaaactcaaatgcgtcggcgtgcggccgctgccagttgcatttactgctgctc ccgcttaaagagcagaaactcattggctgtcctagggtacctggacgctccttgtttactgctgctcccgcttaaagagcagaaactcatggctggtgccgcaccgg taagcagaactagaggtggcaccggtttgattgcacagctttactgctgctcccgcttaaagagcagaaactcatgctggcagccggggtaccgcaggcaagtcgct ttactgctgctcccgcttaaagagcagaaactcaatggaatcacggcggccgcccgtatctcatcctttactgctgctcccgcttaaagagcagaaactcaaatgcg tcggcgtgcggccgctgccagttgcatttactgctgctcccgcttaaagagcagaaactcattggctgtcctagggtacctggacgctccttgtttactgctgctcc cgcttaaagagcagaaactca 10x[b] TALB binding site [b] tcttccgtttccacatct taaagagcagaaactcatcttccgtttccacatcttgctggcagccggggtaccgcaggcaagtcgctaaagagcagaaactcatcttccgtttccacatctatgga atcacggcggccgcccgtatctcatcctaaagagcagaaactcatcttccgtttccacatctaatgcgtcggcgtgcggccgctgccagttgcataaagagcagaaa ctcatcttccgtttccacatctttggctgtcctagggtacctggacgctccttgtaaagagcagaaactcatcttccgtttccacatcttggctggtgccgcaccgg taagcagaactagaggtggcaccggtttgattgcacagctaaagagcagaaactcatcttccgtttccacatcttgctggcagccggggtaccgcaggcaagtcgct aaagagcagaaactcatcttccgtttccacatctatggaatcacggcggccgcccgtatctcatcctaaagagcagaaactcatcttccgtttccacatctaatgcg tcggcgtgcggccgctgccagttgcataaagagcagaaactcatcttccgtttccacatctttggctgtcctagggtacctggacgctccttgtaaagagcagaaac tcatcttccgtttccacatct l 1 tggctggtgccgcaccggtaagcagaggatct l 2 Sequence between binding sites and Pmin. Sequence between binding sites and PCMV. tggctggtgccgcaccggtaagcagaggatcctgtacgggccagatatacgcgttg l 3 ctcgagggcaatccggtactgttggtaaagccaccgaattc Sequence between Pmin and ozak sequence. l 4 Sequence between PCMV and ozak sequence. aagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattc TALA TALEN1257 from aa 2-804; Addgene plasmid gactacaaagaccatgacggtgattataaagatcatgacatcgattacaaggatgacgatgacaagatggcccccaagaagaagaggaaggtgggcattcaccgcgg ggtacctatggtggacttgaggacactcggttattcgcaacagcaacaggagaaaatcaagcctaaggtcaggagcaccgtcgcgcaacaccacgaggcgcttgtgg ggcatggcttcactcatgcgcatattgtcgcgctttcacagcaccctgcggcgcttgggacggtggctgtcaaataccaagatatgattgcggccctgcccgaagcc acgcacgaggcaattgtaggggtcggtaaacagtggtcgggagcgcgagcacttgaggcgctgctgactgtggcgggtgagcttagggggcctccgctccagctcga caccgggcagctgctgaagatcgcgaagagagggggagtaacagcggtagaggcagtgcacgcctggcgcaatgcgctcaccggggcccccttgaacctgaccccag accaggtagtcgcaatcgcgtcaaacggagggggaaagcaagccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacggccttacaccggagcaa gtcgtggccattgcaagcaatgggggtggcaaacaggctcttgagacggttcagagacttctcccagttctctgtcaagcccacgggctgactcccgatcaagttgt agcgattgcgtcgaacattggagggaaacaagcattggagactgtccaacggctccttcccgtgttgtgtcaagcccacggtttgacgcctgcacaagtggtcgcca tcgccagccatgatggcggtaagcaggcgctggaaacagtacagcgcctgctgcctgtactgtgccaggatcatggactgaccccagaccaggtagtcgcaatcgcg tcaaacggagggggaaagcaagccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacggccttacaccggagcaagtcgtggccattgcaaataa taacggtggcaaacaggctcttgagacggttcagagacttctcccagttctctgtcaagcccacgggctgactcccgatcaagttgtagcgattgcgtcgcatgacg gagggaaacaagcattggagactgtccaacggctccttcccgtgttgtgtcaagcccacggtttgacgcctgcacaagtggtcgccatcgcctcgaatggcggcggt aagcaggcgctggaaacagtacagcgcctgctgcctgtactgtgccaggatcatggactgaccccagaccaggtagtcgcaatcgcgaacaataatgggggaaagca agccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacggccttacaccggagcaagtcgtggccattgcatcccacgacggtggcaaacaggctc ttgagacggttcagagacttctcccagttctctgtcaagcccacgggctgactcccgatcaagttgtagcgattgcgtccaacggtggagggaaacaagcattggag actgtccaacggctccttcccgtgttgtgtcaagcccacggtttgacgcctgcacaagtggtcgccatcgccagccatgatggcggtaagcaggcgctggaaacagt acagcgcctgctgcctgtactgtgccaggatcatggactgaccccagaccaggtagtcgcaatcgcgtcacatgacgggggaaagcaagccctggaaaccgtgcaaa ggttgttgccggtcctttgtcaagaccacggccttacaccggagcaagtcgtggccattgcatcccacgacggtggcaaacaggctcttgagacggttcagagactt ctcccagttctctgtcaagcccacgggctgactcccgatcaagttgtagcgattgcgaataacaatggagggaaacaagcattggagactgtccaacggctccttcc cgtgttgtgtcaagcccacggtttgacgcctgcacaagtggtcgccatcgccagccatgatggcggtaagcaggcgctggaaacagtacagcgcctgctgcctgtac tgtgccaggatcatggactgacacccgaacaggtggtcgccattgcttctaatgggggaggacggccagccttggagtccatcgtagcccaattgtccaggcccgat cccgcgttggctgcgttaacgaatgaccatctggtggcgttggcatgtcttggtggacgacccgcgctcgatgcagtcaaaaagggtctgcctcatgctcccgcatt gatcaaaagaaccaaccggcggattcccgagagaacttcccatcgagtcgcgggatcc DYDHDGDYDHDIDYDDDDMAPRVGIHRGVPMVDLRTLGYSQQQQEIPVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVYQDMIAALPEA THEAIVGVGQWSGARALEALLTVAGELRGPPLQLDTGQLLIARGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGQALETVQRLLPVLCQDHGLTPEQ VVAIASNGGGQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGQALETVQRLLPVLCQDHGLTPDQVVAIA SNGGGQALETVQRLLPVLCQDHGLTPEQVVAIANNNGGQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGQALETVQRLLPVLCQAHGLTPAQVVAIASNGGG QALETVQRLLPVLCQDHGLTPDQVVAIANNNGGQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGQALE TVQRLLPVLCQAHGLTPAQVVAIASHDGGQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGQALETVQRL LPVLCQAHGLTPDQVVAIANNNGGQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPD PALAALTNDHLVALACLGGRPALDAVGLPHAPALIRTNRRIPERTSHRVAGS TALB TALEN1297 from aa 2-804; Addgene plasmid gactacaaagaccatgacggtgattataaagatcatgacatcgattacaaggatgacgatgacaagatggcccccaagaagaagaggaaggtgggcattcaccgcgg ggtacctatggtggacttgaggacactcggttattcgcaacagcaacaggagaaaatcaagcctaaggtcaggagcaccgtcgcgcaacaccacgaggcgcttgtgg ggcatggcttcactcatgcgcatattgtcgcgctttcacagcaccctgcggcgcttgggacggtggctgtcaaataccaagatatgattgcggccctgcccgaagcc

11 acgcacgaggcaattgtaggggtcggtaaacagtggtcgggagcgcgagcacttgaggcgctgctgactgtggcgggtgagcttagggggcctccgctccagctcga caccgggcagctgctgaagatcgcgaagagagggggagtaacagcggtagaggcagtgcacgcctggcgcaatgcgctcaccggggcccccttgaacctgaccccag accaggtagtcgcaatcgcgtcacatgacgggggaaagcaagccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacggccttacaccggagcaa gtcgtggccattgcaagcaatgggggtggcaaacaggctcttgagacggttcagagacttctcccagttctctgtcaagcccacgggctgactcccgatcaagttgt agcgattgcgtccaacggtggagggaaacaagcattggagactgtccaacggctccttcccgtgttgtgtcaagcccacggtttgacgcctgcacaagtggtcgcca tcgccagccatgatggcggtaagcaggcgctggaaacagtacagcgcctgctgcctgtactgtgccaggatcatggactgaccccagaccaggtagtcgcaatcgcg tcacatgacgggggaaagcaagccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacggccttacaccggagcaagtcgtggccattgcaaataa taacggtggcaaacaggctcttgagacggttcagagacttctcccagttctctgtcaagcccacgggctgactcccgatcaagttgtagcgattgcgtccaacggtg gagggaaacaagcattggagactgtccaacggctccttcccgtgttgtgtcaagcccacggtttgacgcctgcacaagtggtcgccatcgcctcgaatggcggcggt aagcaggcgctggaaacagtacagcgcctgctgcctgtactgtgccaggatcatggactgaccccagaccaggtagtcgcaatcgcgtcaaacggagggggaaagca agccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacggccttacaccggagcaagtcgtggccattgcatcccacgacggtggcaaacaggctc ttgagacggttcagagacttctcccagttctctgtcaagcccacgggctgactcccgatcaagttgtagcgattgcgtcgcatgacggagggaaacaagcattggag actgtccaacggctccttcccgtgttgtgtcaagcccacggtttgacgcctgcacaagtggtcgccatcgcctccaatattggcggtaagcaggcgctggaaacagt acagcgcctgctgcctgtactgtgccaggatcatggactgaccccagaccaggtagtcgcaatcgcgtcacatgacgggggaaagcaagccctggaaaccgtgcaaa ggttgttgccggtcctttgtcaagaccacggccttacaccggagcaagtcgtggccattgcaagcaacatcggtggcaaacaggctcttgagacggttcagagactt ctcccagttctctgtcaagcccacgggctgactcccgatcaagttgtagcgattgcgtccaacggtggagggaaacaagcattggagactgtccaacggctccttcc cgtgttgtgtcaagcccacggtttgacgcctgcacaagtggtcgccatcgccagccatgatggcggtaagcaggcgctggaaacagtacagcgcctgctgcctgtac tgtgccaggatcatggactgacacccgaacaggtggtcgccattgcttctaatgggggaggacggccagccttggagtccatcgtagcccaattgtccaggcccgat cccgcgttggctgcgttaacgaatgaccatctggtggcgttggcatgtcttggtggacgacccgcgctcgatgcagtcaaaaagggtctgcctcatgctcccgcatt gatcaaaagaaccaaccggcggattcccgagagaacttcccatcgagtcgcgggatcc DYDHDGDYDHDIDYDDDDMAPRVGIHRGVPMVDLRTLGYSQQQQEIPVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVYQDMIAALPEA THEAIVGVGQWSGARALEALLTVAGELRGPPLQLDTGQLLIARGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGQALETVQRLLPVLCQDHGLTPEQ VVAIASNGGGQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGQALETVQRLLPVLCQDHGLTPDQVVAIA SHDGGQALETVQRLLPVLCQDHGLTPEQVVAIANNNGGQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGQALETVQRLLPVLCQAHGLTPAQVVAIASNGGG QALETVQRLLPVLCQDHGLTPDQVVAIASNGGGQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGQALE TVQRLLPVLCQAHGLTPAQVVAIASNIGGQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGQALETVQRL LPVLCQAHGLTPDQVVAIASNGGGQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPD PALAALTNDHLVALACLGGRPALDAVGLPHAPALIRTNRRIPERTSHRVAGS RAB Addgene plasmid ggcggtggtgctttgtctcctcagcactctgctgtcactcaaggaagtatcatcaagaacaaggagggcatggatgctaagtcactaactgcctggtcccggacact ggtgaccttcaaggatgtatttgtggacttcaccagggaggagtggaagctgctggacactgctcagcagatcgtgtacagaaatgtgatgctggagaactataaga acctggtttccttgggttatcagcttactaagccagatgtgatcctccggttggagaagggagaagagccctggctggtggagagagaaattcaccaagagacccat cctgattcagagactgcatttgaaatcaaatcatcagtt GGGALSPQHSAVTQGSIINEGMDASLTAWSRTLVTFDVFVDFTREEWLLDTAQQIVYRNVMLENYNLVSLGYQLTPDVILRLEGEEPWLVEREIHQETH PDSETAFEISSV VP16 39 gcacccccgaccgatgtcagcctgggggacgagctccacttagacggcgaggacgtggcgatggcgcatgccgacgcgctagacgatttcgatctggacatgttggg ggacggggattccccgggtccgggatttaccccccacgactccgccccctacggcgctctggatatggccgacttcgagtttgagcagatgtttaccgatgcccttg gaattgacgagtacggtggg APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG NLS gatccaaaaaagaagagaaaggta DPRV t2a peptide 42 SV40 large T-antigen nuclear localization sequence agagccgaggggagaggaagtcttctgacctgcggagacgtcgaagagaatcctggaccc RAEGRGSLLTCGDVEENPGP mcitrine 40 gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcga tgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgatgtgcttcg cccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaag acccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagta caactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcg ccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaag cgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag VSGEELFTGVVPILVELDGDVNGHFSVSGEGEGDATYGLTLFICTTGLPVPWPTLVTTFGYGLMCFARYPDHMQHDFFSAMPEGYVQERTIFFDDGNY TRAEVFEGDTLVNRIELGIDFEDGNILGHLEYNYNSHNVYIMADQNGIVNFIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSDPNE RDHMVLLEFVTAAGITLGMDELY TagBFP ptagbfp-n (Evrogen) agcgagctgattaaggagaacatgcacatgaagctgtacatggagggcaccgtggacaaccatcacttcaagtgcacatccgagggcgaaggcaagccctacgaggg cacccagaccatgagaatcaaggtggtcgagggcggccctctccccttcgccttcgacatcctggctactagcttcctctacggcagcaagaccttcatcaaccaca cccagggcatccccgacttcttcaagcagtccttccctgagggcttcacatgggagagagtcaccacatacgaagacgggggcgtgctgaccgctacccaggacacc agcctccaggacggctgcctcatctacaacgtcaagatcagaggggtgaacttcacatccaacggccctgtgatgcagaagaaaacactcggctgggaggccttcac cgagacgctgtaccccgctgacggcggcctggaaggcagaaacgacatggccctgaagctcgtgggcgggagccatctgatcgcaaacatcaagaccacatatagat ccaagaaacccgctaagaacctcaagatgcctggcgtctactatgtggactacagactggaaagaatcaaggaggccaacaacgagacctacgtcgagcagcacgag gtggcagtggccagatactgcgacctccctagcaaactggggcacaagcttaat SELIENMHMLYMEGTVDNHHFCTSEGEGPYEGTQTMRIVVEGGPLPFAFDILATSFLYGSTFINHTQGIPDFFQSFPEGFTWERVTTYEDGGVLTATQDT SLQDGCLIYNVIRGVNFTSNGPVMQTLGWEAFTETLYPADGGLEGRNDMALLVGGSHLIANITTYRSPANLMPGVYYVDYRLERIEANNETYVEQHE VAVARYCDLPSLGHLN fluc Firefly luciferase from the plasmid vector pgl4.16 gaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcc cggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctga atacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatc tacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctacc gatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacg acttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgct tgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcac cacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgc tggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggag

12 gtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaa gcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtg gccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggac gagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatctt cgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggact atgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgc gagattctcattaaggccaagaagggcggcaagatcgccgtgaattctgcttgcaagaactggttcagtagcttaagccactttgtgatccaccttaacagccacgg cttccctcccgaggtggaggagcaggccgccggcaccctgcccatgagctgcgcccaggagagcggcatggatagacaccctgctgcttgcgccagcgccaggatca acgtc EDANIGPAPFYPLEDGTAGEQLHAMRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDI YNERELLNSMGISQPTVVFVSGLQILNVQLPIIQIIIMDSTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDTIALIMNSSGSTGLPGVALPHRTA CVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYIQSALLVPTLFSFFASTLIDYDLSNLHEIASGGAPLSE VGEAVARFHLPGIRQGYGLTETTSAILITPEGDDPGAVGVVPFFEAVVDLDTGTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDDGWLHSGDIAYWDED EHFFIVDRLSLIYGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGTMTEEIVDYVASQVTTALRGGVVFVDEVPGLTGLDARIR EILIAGGIAVNSACNWFSSLSHFVIHLNSHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV E Erythromycin inducible protein 25 ccccgccccaagctcaagtccgatgacgaggtactcgaggccgccaccgtagtgctgaagcgttgcggtcccatagagttcacgctcagcggagtagcaaaggaggt ggggctctcccgcgcagcgttaatccagcgcttcaccaaccgcgatacgctgctggtgaggatgatggagcgcggcgtcgagcaggtgcggcattacctgaatgcga taccgataggcgcagggccgcaagggctctgggaatttttgcaggtgctcgttcggagcatgaacactcgcaacgacttctcggtgaactatctcatctcctggtac gagctccaggtgccggagctacgcacgcttgcgatccagcggaaccgcgcggtggtggaggggatccgcaagcgactgcccccaggtgctcctgcggcagctgagtt gctcctgcactcggtcatcgctggcgcgacgatgcagtgggccgtcgatccggatggtgagctagctgatcatgtgctggctcagatcgctgccatcctgtgtttaa tgtttcccgaacacgacgatttccaactcctccaggcacatgcgtccgcgtacagc PRPLSDDEVLEAATVVLRCGPIEFTLSGVAEVGLSRAALIQRFTNRDTLLVRMMERGVEQVRHYLNAIPIGAGPQGLWEFLQVLVRSMNTRNDFSVNYLISWY ELQVPELRTLAIQRNRAVVEGIRRLPPGAPAAAELLLHSVIAGATMQWAVDPDGELADHVLAQIAAILCLMFPEHDDFQLLQAHASAYS PIP Pristinamycin inducible protein 26 agtcgaggagaggtgcgcatggcgaaggcagggcgggaggggccgcgggacagcgtgtggctgtcgggggaggggcggcgcggcggtcgccgtggggggcagccgtc cgggctcgaccgggaccggatcaccggggtcaccgtccggctgctggacacggagggcctgacggggttctcgatgcgccgcctggccgccgagctgaacgtcaccg cgatgtccgtgtactggtacgtcgacaccaaggaccagttgctcgagctcgccctggacgccgtcttcggcgagctgcgccacccggacccggacgccgggctcgac tggcgcgaggaactgcgggccctggcccgggagaaccgggcgctgctggtgcgccacccctggtcgtcccggctggtcggcacctacctcaacatcggcccgcactc gctggccttctcccgcgcggtgcagaacgtcgtgcgccgcagcgggctgcccgcgcaccgcctgaccggcgccatctcggccgtcttccagttcgtctacggctacg gcaccatcgagggccgcttcctcgcccgggtggcggacaccgggctgagtccggaggagtacttccaggactcgatgaccgcggtgaccgaggtgccggacaccgcg ggcgtcatcgaggacgcgcaggacatcatggcggcccggggcggcgacaccgtggcggagatgctggaccgggacttcgagttcgccctcgacctgctcgtcgcggg catcgacgcgatggtcgaacaggcctccgcgtacagc SRGEVRMAAGREGPRDSVWLSGEGRRGGRRGGQPSGLDRDRITGVTVRLLDTEGLTGFSMRRLAAELNVTAMSVYWYVDTDQLLELALDAVFGELRHPDPDAGLD WREELRALARENRALLVRHPWSSRLVGTYLNIGPHSLAFSRAVQNVVRRSGLPAHRLTGAISAVFQFVYGYGTIEGRFLARVADTGLSPEEYFQDSMTAVTEVPDTA GVIEDAQDIMAARGGDTVAEMLDRDFEFALDLLVAGIDAMVEQASAYS 8x[etr] E binding site [etr] gattgaatataaccgacgtgactgttacatttagg 25 agcttcgaagtttaaacgattgaatataaccgacgtgactgttacatttagggattgaatataaccgacgtgactgttacatttagggattgaatataaccgacgtg actgttacatttagggattgaatataaccgacgtgactgttacatttagggaaaacgattgaatataaccgacgtgactgttacatttagggattgaatataaccga cgtgactgttacatttagggattgaatataaccgacgtgactgttacatttagggattgaatataaccgacgtgactgttacatttagggatatcctgcagggaatt 3x[pir] PIP binding site [pir] tacggtgtacgggaagatactcgtacaccgtacaagagattcccatacgctgtacagcgctatttcg 26 cctgcagggtacggtgtacgggaagatactcgtacaccgtacaagagattcccatacgctgtacagcgctatttcgtacggtgtacgggaagatactcgtacaccgt acaagagattcccatacgctgtacagcgctatttcgtacggtgtacgggaagatactcgtacaccgtacaagagattcccatacgctgtacagcgctatttcgatcg aatt irfp670 GenBank: AGN atggccagaaaggtggacctgaccagctgcgacagagagcccatccacatccccggcagcatccagccttgtggctgtctgctggcctgtgatgctcaggctgtgcg gatcaccagaatcaccgagaacgctggcgccttcttcggacgggaaacacctagagtgggagagctgctggccgactacttcggcgagacagaagcccacgccctga gaaatgccctggcccagagcagcgaccctaagagacctgccctgatcttcggctggcgggatggactgaccggccggacctttgacatcagcctgcacagacacgac ggcaccagcatcatcgagttcgagcctgccgccgctgagcaggctgacaatcctctgagactgacccggcagatcattgcccggaccaaagagctgaagtccctgga agagatggccgccagagtgcctagatacctccaggccatgctgggctaccacagagtgatgctgtacagattcgccgacgacggcagcggcatggtcatcggagagg ccaagagaagcgacctggaatccttcctgggccagcacttccctgccagcctggtgcctcagcaggccagactgctgtacctgaagaacgccatccgggtggtgtcc gacagcagaggcatcagcagcagaatcgtgcccgagcacgacgccagcggagctgcactggatctgagctttgcccacctgagatccatcagcccttgccacctgga atttctgcggaacatgggcgtgtccgccagcatgtccctgtccatcatcatcgatggcaccctgtggggcctgatcatctgccaccactacgagcccagagccgtgc ccatggctcagagggtggccgccgaaatgttcgccgacttcctgtccctgcacttcacagccgcccaccaccagagataa MARVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAGAFFGRETPRVGELLADYFGETEAHALRNALAQSSDPRPALIFGWRDGLTGRTFDISLHRHD GTSIIEFEPAAAEQADNPLRLTRQIIARTELSLEEMAARVPRYLQAMLGYHRVMLYRFADDGSGMVIGEARSDLESFLGQHFPASLVPQQARLLYLNAIRVVS DSRGISSRIVPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYEPRAVPMAQRVAAEMFADFLSLHFTAAHHQR mcherry mcherry fluorescent protein from the plasmid vector pmcherry-c1 (Clontech) atggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgaggg cgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgt acggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggc ggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaa gaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacg acgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacacc atcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaag MVSGEEDNMAIIEFMRFVHMEGSVNGHEFEIEGEGEGRPYEGTQTALVTGGPLPFAWDILSPQFMYGSAYVHPADIPDYLLSFPEGFWERVMNFEDG GVVTVTQDSSLQDGEFIYVLRGTNFPSDGPVMQTMGWEASSERMYPEDGALGEIQRLLDGGHYDAEVTTYAPVQLPGAYNVNILDITSHNEDYT IVEQYERAEGRHSTGGMDELY rluc Renilla luciferase from the plasmid vector phrl-t (Promega) atggcttccaaggtgtacgaccccgagcaacgcaaacgcatgatcactgggcctcagtggtgggctcgctgcaagcaaatgaacgtgctggactccttcatcaacta ctatgattccgagaagcacgccgagaacgccgtgatttttctgcatggtaacgctgcctccagctacctgtggaggcacgtcgtgcctcacatcgagcccgtggcta gatgcatcatccctgatctgatcggaatgggtaagtccggcaagagcgggaatggctcatatcgcctcctggatcactacaagtacctcaccgcttggttcgagctg ctgaaccttccaaagaaaatcatctttgtgggccacgactggggggcttgtctggcctttcactactcctacgagcaccaagacaagatcaaggccatcgtccatgc tgagagtgtcgtggacgtgatcgagtcctgggacgagtggcctgacatcgaggaggatatcgccctgatcaagagcgaagagggcgagaaaatggtgcttgagaata acttcttcgtcgagaccatgctcccaagcaagatcatgcggaaactggagcctgaggagttcgctgcctacctggagccattcaaggagaagggcgaggttagacgg cctaccctctcctggcctcgcgagatccctctcgttaagggaggcaagcccgacgtcgtccagattgtccgcaactacaacgcctaccttcgggccagcgacgatct gcctaagatgttcatcgagtccgaccctgggttcttttccaacgctattgtcgagggagctaagaagttccctaacaccgagttcgtgaaggtgaagggcctccact tcagccaggaggacgctccagatgaaatgggtaagtacatcaagagcttcgtggagcgcgtgctgaagaacgagcag MASVYDPEQRRMITGPQWWARCQMNVLDSFINYYDSEHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGSGSGNGSYRLLDHYYLTAWFEL

13 LNLPIIFVGHDWGACLAFHYSYEHQDIAIVHAESVVDVIESWDEWPDIEEDIALISEEGEMVLENNFFVETMLPSIMRLEPEEFAAYLEPFEGEVRR PTLSWPREIPLVGGPDVVQIVRNYNAYLRASDDLPMFIESDPGFFSNAIVEGAFPNTEFVVGLHFSQEDAPDEMGYISFVERVLNEQ Legend: amino acid sequences are shaded gray; yellow and green shades indicate individual binding sites.

14 Supplementary Table 3 Amount of transfected plasmids for TALE-based genetic switches in each well of the 8, 12, 24 or 96-well plates. For the mutual repressor switch, constructs with CMV promoters were used (Fig. 1), while versions with either the CMV promoter (Fig. 4, Supplementary Fig. 6) or the minimal promoter (Fig. 7, Fig. 8 and Fig. 9) were tested for the competitive feedback switch. In the 96-well plates, two versions (A and B) of the switch with the firefly luciferase reporter were tested to verify the high and low levels of expression of both possible states. The reporters for the confocal microscopy and flow cytometry experiments (8-well and 12- or 24-well plates, respectively) were mcitrine linked to TALA:RAB and TagBFP linked to TALB:RAB. Plasmids Mutual repressor switch 8-well [ng] 8-well [ng] Competitive feedback switch 12-well [ng] 24-well [ng] 96-well [ng] version A 96-well [ng] version B 10x[a]_l 4_[promoter]_l 2_TALB:RAB:t2A:reporter / 15 10x[a]_l 4_[promoter]_l 2_TALB:RAB / / / / 15 / 10x[a]_l 4_[promoter]_l 2_TALA:VP16 / ,5 2,5 10x[b]_l 4_[promoter]_l 2_TALA:RAB:t2A:reporter / 10x[b]_l 4_[promoter]_l 2_TALA:RAB / / / / / 15 10x[b]_l 4_[promoter]_l 2_TALB:VP16 / ,5 2,5 3x[pir]_l 4_[CMV]_l 2_TALB:RAB x[pir]_l 4_[CMV]_l 2_TALA:VP16 / x[etr]_l 4_[CMV]_l 2_TALA:RAB x[etr]_l 4_[CMV]_l 2_TALB:VP16 / [CMV]_l 2_PIP:RAB [CMV]_l 2_E:RAB transfection control [CMV]_mCherry / / / / transfection control [CMV]_iRFP670 / / / / transfection control [T]_rLuc / / / / 5 5

15 Supplementary Table 4 Labels and descriptions of chemical species used in deterministic and stochastic models. Label T T V T T A T AV T A T CV T B T BV T B T DV BFP mct PIP E PI ER PI_PIP ER_E PMIN T _PMIN T V _PMIN T V _T V _PMIN T _T _PMIN T _T V _PMIN PCMV T _PCMV T V _PCMV T V _T V _PCMV T_T_PCMV T _T _PCMV T _T V _PCMV T _T_PCMV PIP _PCMV PIP (PCMV PIP ) E _PCMV E (PCMV E ) Chemical species TALE repressor TALE activator TALE DNA binding domain TALA:RAB repressor TALA:VP16 activator TALA DNA binding domain TALC:VP16 activator TALB:RAB repressor TALB:VP16 activator TALB DNA binding domain TALD:VP16 activator TagBFP fluorescent protein reporter mcitrine fluorescent protein reporter pristinamycin-inducible repressor (PIP:RAB) erythromycin-inducible repressor (E:RAB) pristinamycin inducer molecule erythromycin inducer molecule PIP-pristinamycin complex E-erythromycin complex minimal promoter minimal promoter (TALE repressor bound) minimal promoter (TALE activator bound) minimal promoter (two TALE activators bound) minimal promoter (two TALE repressors bound) minimal promoter (combination of a bound TALE repressor and a bound TALE activator) CMV promoter CMV promoter (TALE repressor bound) CMV promoter (TALE activator bound) CMV promoter (two TALE activators bound) CMV promoter (two TALE DNA binding domains bound) CMV promoter (two TALE repressors bound) CMV promoter (combination of a bound TALE repressor and a bound TALE activator) CMV promoter (combination of a bound TALE repressor and a bound TALE DNA binding domain) CMV promoter (PIP:RAB bound) CMV promoter (E:RAB bound)

16 Supplementary Table 5 Parameter values used by the deterministic ODE model in the mutual repressor switch simulations. The k b values were chosen depending on the percentage of incomplete repression (0% or 5%) in individual simulations (Fig. 1c). Initial protein concentrations in all simulations are equal to zero. Parameter label Parameter description Value n 1, n 2, n 3, n 4 Hill coefficients 1.0 or 2.0 1, 2, 3, 4 association constants 1 k BFP, k mct protein production rates of fluorescent reporters 100 h -1 k bbfp, k bmct protein production rates of fluorescent reporters on account of incomplete repression 0 or 5 h -1 k 1BR, k 2AR protein production rates of TALE repressors from constructs No. 17 and No. 18 (Supplementary Table 1) 180 h -1 k b1br, k b2ar protein production rates of TALE repressors from constructs No. 17 and No. 18 (Supplementary Table 1) on account of incomplete repression 0 or 9 h -1 k 3BR, k 4AR protein production of TALE repressors from constructs No. 11 and No. 13 (Supplementary Table 1) 30 h -1 k b3br, k b4ar protein production of TALE repressors from constructs No. 11 and No. 13 (Supplementary Table 1) on account of incomplete repression 0 or 1.5 h -1 k PIP, k E protein production rates of PIP:RAB and E:RAB inducible repressors 150 h -1 q i protein degradation rates 0.1 h -1

17 Supplementary Table 6 Values used by the deterministic ODE model in the competitive feedback switch simulations. The k b values were chosen depending on the percentage of incomplete repression or unprompted activation (0% or 1%) in individual simulations (Supplementary Fig. 3). Initial protein concentrations in all simulations are equal to zero. Parameter label Parameter description Value 1, 2, 3, 4, 5, 6 association constants 1 k BFP, k mct protein production rates of fluorescent reporters 100 h -1 k bbfp, k bmct protein production rates of fluorescent reporters on account of incomplete repression 0 or 1 h -1 k 1BR, k 2AR protein production rates of TALE repressors from constructs No. 21 and No. 22 (Supplementary Table 1) 180 h -1 k b1br, k b2ar protein production rates of TALE repressors from constructs No. 21 and No. 22 (Supplementary Table 1) on account of incomplete repression 0 or 1.8 h -1 k 1AVP, k 2BVP protein production rates of TALE activators from constructs No. 27 and No. 28 (Supplementary Table 1) 30 h -1 k b1avp, k b2bvp protein production rates of TALE activators from constructs No. 27 and No. 28 (Supplementary Table 1) on account of incomplete repression 0 or 0.3 h -1 k 3BR, k 4AR protein production rates of TALE repressors from constructs No. 11 and No. 13 (Supplementary Table 1) 60 h -1 k b3br, k b4ar protein production rates of TALE repressors from constructs No. 11 and No. 13 (Supplementary Table 1) on account of incomplete repression 0 or 6 h -1 k 3AVP, k 4BVP protein production rates of TALE activators from constructs No. 12 and No. 14 (Supplementary Table 1) 60 h -1 k b3avp, k b4bvp protein production rates of TALE activators from constructs No. 12 and No. 14 (Supplementary Table 1) on account of incomplete repression 0 or 6 h -1 k PIP, k E protein production rates of PIP and E inducible repressors 150 h -1 q i protein degradation rates 0.1 h -1

18 Supplementary Table 7 Parameters used in the stochastic models and their respective values and references. The parameter values are consistent for the comparison of the switch topology models. The estimate of the parameter order of magnitude is based on 3, p. 6, Table 2.1 and normalized to 1 nmol/s. N/A indicates unitless parameters. Parameter label Parameter description Value Unit Reference protein production constant for CMV promoters r CMV 10-4 nmol/s 3 (including mrna nuclear export) protein production constant for minimal promoters with r VV 10-4 nmol/s 3 two bound activators protein production constant for minimal promoters with a r V 10-6 nmol/s 3 bound activator and a repressor N/A estimated from V CMV CMV promoter activation constant for bound activators 10.0 experimental data CMV promoter activation constant for a bound activator N/A estimated from V CMV 2.0 and a repressor experimental data leakage percentage (incomplete repression of the CMV N/A estimated from l CMV 0.01 promoter) experimental data 3 k PI pristinamycin-pip binding constant 1.0 nmol/s 3 k ER erythromycin-e binding constant 1.0 nmol/s 3 k T TALE regulator-dna binding constant 1.0 nmol/s weighing factor for stronger effect of repression over N/A estimated from α T 3.05 activation experimental data 3 k PIP PIP-DNA binding constant 1.0 nmol/s 3 k E E-DNA binding constant 1.0 nmol/s k T TALE-DNA association constant 1.0 nmol/s 3 q T TALE-DNA dissociation constant nmol/s q PIP PIP-DNA dissociation constant 10-4 nmol/s equal to q T q E E-DNA dissociation constant 10-4 nmol/s equal to q T d T TALE regulator degradation constant nmol/s d PIP PIP:RAB degradation constant 10-5 nmol/s equal to d T d E E.RAB degradation constant 10-5 nmol/s equal to d T d mct mcitrine fluorescent protein degradation constant 10-5 nmol/s equal to d T d BFP Blue fluorescent protein degradation constant 10-5 nmol/s equal to d T d RFP Red fluorescent protein degradation constant 10-5 nmol/s equal to d T RLU/ estimated from f RLU Reporter concentration-rlu conversion factor nmol experimental data

19 Supplementary Table 8 Constructs included in the stochastic model of the competitive feedback switch topology based on constitutive promoters. Construct label Construct name Construct description PCMV A0 [a]_[cmv]_talb:rab:t2a:bfp Supplementary Table 1; No. 18 PCMV A1 [a]_[cmv]_tala:vp16 Supplementary Table 1; No. 19 PCMV B0 [b]_[cmv]_tala:rab:t2a:mcit Supplementary Table 1; No. 17 PCMV B1 [b]_[cmv]_talb:vp16 Supplementary Table 1; No. 20 PCMV PIP0 [pir]_[cmv]_talb:rab Supplementary Table 1; No. 11 PCMV PIP1 [pir]_[cmv]_tala:vp16 Supplementary Table 1; No. 12 PCMV E0 [etr]_[cmv]_tala:rab Supplementary Table 1; No. 13 PCMV E1 [etr]_[cmv]_talb:vp16 Supplementary Table 1; No. 14

20 Supplementary Table 9 Reaction set for the stochastic model of the competitive feedback switch topology based on constitutive promoters. The chemical species used in the model are listed in Supplementary Table 4. The constructs are labeled in the following manner: promoter binding sites_operon. PCMV represents a constitutive CMV promoter. The binding sites annotated A, B represent the TALA and TALB DNA-binding sites, respectively, and the binding sites annotated PIP, E represent the PIP:RAB and E:RAB DNA-binding sites, respectively. Operons with the notation 0 encode the TALE repressors (and optionally reporter proteins), while operons with the notation 1 encode the TALE activators. For a detailed description of the PCMV A0, PCMV A1, PCMV B0, PCMV B1, PCMV PIP0, PCMV PIP1, PCMV E0 and PCMV E1 constructs included in the model, see Supplementary Table 8. Reaction Reaction description PCMV A0 + T A + T A Binding and dissociation of two TALA PCMV A0 + T A + T A qt T A _T A _PCMV A0 repressors to the PCMV A0 construct PCMV A0 + T A + T AV Binding and dissociation of a TALA repressor PCMV A0 + T A + T AV qt T A _T AV _PCMV A0 and a TALA activator to the PCMV A0 construct PCMV A0 + T AV + T AV kt T AV _T AV _PCMV A0 Binding and dissociation of two TALA PCMV A0 + T AV + T AV qt T AV _T AV _PCMV A0 activators to the PCMV A0 construct PCMV A1 + T A + T A Binding and dissociation of two TALA PCMV A1 + T A + T A qt T A _T A _PCMV A1 repressors to the PCMV A1 construct PCMV A1 + T A + T AV Binding and dissociation of a TALA repressor PCMV A1 + T A + T AV qt T A _T AV _PCMV A1 and a TALA activator to the PCMV A1 construct PCMV A1 + T AV + T AV kt T AV _T AV _PCMV A1 Binding and dissociation of two TALA PCMV A1 + T AV + T AV qt T AV _T AV _PCMV A1 activators to the PCMV A1 construct PCMV B0 + T B + T B Binding and dissociation of two TALB PCMV B0 + T B + T B qt T B _T B _PCMV B0 repressors to the PCMV B0 construct PCMV B0 + T B + T BV Binding and dissociation of a TALB repressor PCMV B0 + T B + T BV qt T B _T BV _PCMV B0 and a TALB activator to the PCMV B0 construct PCMV B0 + T BV + T BV kt T BV _T BV _PCMV B0 Binding and dissociation of two TALB PCMV B0 + T BV + T BV qt T BV _T BV _PCMV B0 activators to the PCMV B0 construct PCMV B1 + T B + T B Binding and dissociation of two TALB PCMV B1 + T B + T B qt T B _T B _PCMV B1 repressors to the PCMV B1 construct PCMV B1 + T B + T BV Binding and dissociation of a TALB repressor PCMV B1 + T B + T BV qt T B _T BV _PCMV B1 and a TALB activator to the PCMV B1 construct PCMV B1 + T BV + T BV kt T BV _T BV _PCMV B1 Binding and dissociation of two TALB PCMV B1 + T BV + T BV qt T BV _T BV _PCMV B1 activators to the PCMV B1 construct Production of TALB:RAB and TagBFP from T A _T AV _PCMV A0 CMV T A _T AV _PCMV A0 + T B + BFP the PCMV A0 construct with a bound repressor and a bound activator Production of TALA:VP16 from the PCMV A1 T A _T AV _PCMV A1 CMV T A _T AV _PCMV A1 + T AV construct with a bound repressor and a bound activator T AV _T AV _CMV A0 VCMV T AV _T AV _PCMV A0 + T B + BFP Production of TALB:RAB and TagBFP from the activated PCMV A0 construct T AV _T AV _PCMV A1 VCMV T AV _T AV _PCMV A1 + T AV Production of TALA:VP16 from the activated PCMV A1 construct Production of TALB:RAB and TagBFP from T A _T A _PCMV A0 lcmv T A _T A _PCMV A0 + T B + BFP the repressed PCMV A0 construct on account of incomplete repression Production of TALA:VP16 from the repressed T A _T A _PCMV A1 lcmv T A _T A _PCMV A1 + T AV PCMV A1 construct on account of incomplete repression PCMV A0 PCMV A0 + T B + BFP Production of TALB:RAB and TagBFP from the PCMV A0 construct Production of TALA:VP16 from the PCMV PCMV A1 PCMV A1 + T A1 AV construct Production of TALA:RAB and mcitrine from T B _T BV _PCMV B0 CMV T B _T BV _PCMV B0 + T A + mct the PCMV B0 construct with a bound repressor and a bound activator T B _T BV _PCMV B1 CMV T B _T BV _PCMV B1 + T BV Production of TALB:VP16 from the PCMV B1 construct with a bound repressor and a bound