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	Fig. 1: Schematic diagram of the tPTS strategy to incorporate recombinant and synthetic proteins. The chosen split inteins used in this study, inteins A (CfaDnaE) and B (SspDnaBM86), are indicated by square and round symbols, respectively. Flanking +1, +2, +3 extein residues for each intein are indicated in italics at their respective positions in the top panel. The +1 extein residue (underlined) is a critical requirement for splicing to occur. X denotes that the type of residue at that position is not critical for splicing, although they might affect the kinetics or splicing efficiency.
	Fig. 2: Insertion of recombinantly expressed peptides into the DIII–IV linker of NaV1.5. a Schematic presentation of the strategy to reconstruct full-length NaV1.5 from recombinantly expressed N-/C-terminal fragments (N and C) and a recombinantly expressed peptide corresponding to amino acids 1472 to 1502 of the NaV1.5 DIII–DIV linker (X) in Xenopus laevis oocytes. Inteins A (CfaDnaE) and B (SspDnaBM86) are indicated by square and round symbols, respectively. Note that we cannot exclude the possibility that splicing takes place at a different subcellular location than depicted here. b Representative sodium currents in response to sodium channel activation protocol (see methods; only voltage steps from −50 to +10 mV in 10 mV steps are displayed), demonstrating expression of functional NaV1.5 only in the presence of all three components (N + C + X), along with WT and N1472C channels (the latter mutation was introduced to create an optimized splice site for intein A. c Immunoblots verifying the presence of fully spliced NaV1.5 only when all three components (N + C + X) were co-expressed (using antibody against NaV1.5 C-terminus). NaV1.5 band was not detected when one component was missing (left blot) or when non-splicing mutation (N + C + X mut.) was introduced to prevent splicing (right blot; see Supplementary Fig. 2). Black arrows indicate band positions of the respective constructs (Actual MW of constructs: WT, 227 kDa; C-construct, 79 kDa; C + X, 65 kDa; C-terminal cleavage product, C*, 58 kDa). Note that X and XREC refer to XNav1.5REC in this panel. d Steady-state inactivation and conductance–voltage (G–V) relationships for functional constructs tested. e Comparison of values for half-maximal (in)activation (V50) (values are displayed as mean + /– SD; WT, n = 12; N1472C, n = 14; N + C + XNav1.5REC, n = 16). Significant differences were determined by one-way ANOVA with a Tukey post-hoc test. *p < 0.03 (WT vs. N1472C, p = 0.014; WT vs. N + C + XNav1.5REC, p = 0.012). Source data are provided as a Source data file.
	Fig. 3: Insertion of synthetic peptides into NaV1.5. a Schematic of strategy to reconstruct full-length NaV1.5 from recombinantly expressed N-/C-terminal fragments (N and C) and a synthetic peptide (XNav1.5SYN) in Xenopus laevis oocytes. Inteins A (CfaDnaE) and B (SspDnaBM86) indicated by square and round symbols, respectively. Note: we cannot exclude that splicing takes place at a different subcellular location than depicted. b Peptide XNav1.5SYN sequence corresponding to amino acids replaced in the NaV1.5 DIII–DIV linker and chemical structures of native amino acids and PTM mimics (tAcK/phY) incorporated via chemical synthesis of peptide XNav1.5SYN. The N1472C (underlined) mutation was introduced to optimize splicing (Supplementary Fig. 1). c Immunoblot verifying the presence of fully spliced NaV1.5 only when peptide XNav1.5SYN was co-injected with N and C. Black arrows indicate band positions of the respective constructs (Actual MW of constructs: WT, 227 kDa; C, 79 kDa; C + X, 65 kDa; C-terminal cleavage product, C*, 58 kDa). Band at ~150 kDa is possibly a dimer or aggregate of C. d Representative sodium currents (see Methods; only voltage steps from −50 to +10 mV in 10 mV steps are displayed), demonstrating expression of functional NaV1.5 when Xenopus laevis oocytes expressing N and C constructs were injected with synthetic peptides containing non-modifiable side chains in positions 1479 and 1495 (K1479R and Y1495F, NM), tAcK1479 or phY1495 or both PTM mimics together. e Average current amplitudes recorded at −35 mV from oocytes expressing N and C constructs and injected with synthetic peptide variant NM or phY1495 depicted as a bar plot (mean + /– SD; WT, n = 8; NM, n = 9; phY, n = 8; N + C, n = 6). Currents normalized to mean currents measured from oocytes expressing the full-length WT construct. To ensure adequate control of voltage clamp, [Na+] in the extracellular recording solution was reduced (see Supplementary Fig. 3 for details). f Steady-state inactivation and conductance–voltage (G–V) relationships for PTM-modified/non-modified constructs (values displayed as mean + /– SD; N1472C, n = 15; NM, n = 10; tAcK1479, n = 21; phY1495, n = 19; tAcK1479 + phY1495, n = 14). Source data are provided as a Source data file.
	Fig. 4: Insertion of a recombinantly expressed peptide into GFP expressed in mammalian cells. a Schematic presentation of the strategy applied to reconstitute GFP from recombinantly expressed N-/C-terminal fragments and recombinantly expressed peptide XGFPREC corresponding to amino acids 65–85 of GFP in HEK293 cells. Inteins A (CfaDnaE) and B (SspDnaBM86) are indicated by square and round symbols, respectively. b Bright-field (right panels) and fluorescence (left panels) images of HEK293 cells expressing the indicated constructs. Scale bars: 20 µm. GFP fluorescence was only detected when all three constructs (N + X + C) were co-transfected. GFP fluorescence was not detected when one of the three constructs was absent or when +1 extein residues of each split intein is mutated to alanine (C65A for intein A and S86A for intein B) to prevent splicing.
	Fig. 5: Insertion of synthetic peptides into GFP. a Schematic of strategy applied to reconstitute GFP from recombinantly expressed N-/C-terminal fragments and a synthetic peptide XGFPSYN in HEK293 cells. b Overlay of bright-field and fluorescence images taken of HEK293 cells transfected with only WT GFP, or N and C fragments and squeezed in the presence or absence of peptide XGFPSYN. Scale bars: 50 µm.
	Fig. 6: Insertion of recombinant and synthetic peptides into the P2X2R extracellular domain. a Schematic presentation of the strategy to reconstruct full-length P2X2Rs from recombinantly expressed N-/C-terminal fragments (N and C) and a recombinant (strategy 1, Pept XP2X2REC) or synthetic peptide (strategy 2, Pept XP2X2SYN) into the P2X2R extracellular domain in Xenopus laevis oocytes. Note that a faux transmembrane helix (faux TMD) was engineered into the C-terminal fragment to maintain its native membrane topology (see also Supplementary Fig. 9). Inteins A (CfaDnaE) and B (SspDnaBM86) indicated by square and round symbols, respectively. Peptide X was designed to include a C-terminal ER-targeting KDEL signal sequence, which is excised during the splicing process. However, we cannot exclude the possibility that splicing takes place at a different subcellular location than depicted here. b Representative current traces obtained by application of 30 µM ATP, verifying functional expression only when all three components were recombinantly co-expressed (strategy 1 in a). c Immunoblot of surface-purified proteins. Black arrows on the right indicate band positions of the respective constructs (actual MW of constructs: WT, 53 kDa; C-construct, 97 kDa). Red arrow indicates the band of the spliced full-length receptor. d ATP concentration-response curves (CRCs) for P2X2 WT (black) and K71Q (red) reconstituted from the three co-expressed constructs. Dashed lines represent the CRCs for the respective full-length proteins. e Structures of inserted side chains at position 71 (Lys, Orn, hLys) incorporated via synthetic peptide X (strategy 2 in a). f ATP CRCs indicate successful incorporation of synthetic WT peptide XP2X2SYN (black) with wild-type like ATP sensitivity, while synthetic peptides with Orn (pink) or hLys (blue) resulted in a decrease in ATP sensitivity. Dashed lines indicate ATP CRCs for spliced P2X2R WT (gray) or K71Q (purple) obtained with peptide XP2X2REC. Values in d and f are displayed as mean + /– SD; WT, n = 5; [WT]REC, n = 8; K71Q, n = 7; [K71Q]REC, n = 9; [WT]SYN, n = 7; [K71Orn]SYN, n = 9; [K71hLys]SYN, n = 5). Source data are provided as a Source data file.

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