Table S1.
Plasmids and oligonucleotides used and designed in this study
| Plasmid | Description and cloning strategy | Source |
| pcDNA3.1(+) | Constitutive PhCMV-driven mammalian expression vector (PhCMV-MCS-pAbGH) | Invitrogen |
| pGal4 | Constitutive Gal4 expression vector (PhCMV-Gal4-pAbGH; no. 24345; Addgene) | 20 |
| pSAM200 | Constitutive tTA expression vector (PSV40-tTA-pASV40) | 42 |
| pGL4.23 | Vector encoding a minimal promoter and Luc2 (Pmin-luc2-pASV40) | Promega |
| pSEAP2-Basic | SEAP-containing vector (MCS-SEAP- pASV40) | Clontech |
| pSEAP2-Control | Constitutive SEAP expression vector (PSV40-SEAP-pASV40) | Clontech |
| pTRα | Constitutive hTRα expression vector (PhCMV-hTRα-pAbGH) | Missouri S&T cDNA (NR1A100001) |
| pTSHR | Constitutive hTSHR expression vector (PhCMV-hTSHR-pAbGH) | Missouri S&T cDNA (TSHR000000) |
| pDA134 | PCREm-driven Citrine expression vector (PCREm-Citrine-pASV40) | 40 |
| pDIO2 | Constitutive DIO2 expression vector (PSV40-DIO2-pASV40) | 24 |
| pUC57 | pUC19-derived prokaryotic expression vector | Genscript |
| pWW124 | SCB1-responsive SPA-dependent SEAP expression vector (PSPA-SEAP-pASV40) | 41 |
| pSP15 | pSEAP2-Basic containing CREm (CREm-SEAP-pASV40). OSP19 (5′-acgcgctagcAGCCTGACGTCCGAG AGCCTGACGTCCGAGAGCCTGACGTCCGAGAGCCTGACGTCCGAGATCTCTCGAGGTCGACAGCGGAGACTCTAGAGGGTATATAgaattcacgc-3′) and OSP191 (5′-gcgtgaattcTATATACCCTCTAG AGTCTCCGCTGTCGACCTCGAGAGATCTCGGACGTCAGGCTCTCGGACGTCAGGCTCTCGGACGTCAGGCTCTCGGACGTCAGGCTgctagcgcgt-3′) were annealed, restricted with NheI/EcoRI, and cloned into the corresponding sites (NheI/EcoRI) of pSEAP2-Basic | This work |
| pSP16 | pSEAP2-Basic containing a PCREm-driven SEAP expression unit (PCREm-SEAP-pASV40). pGL4.23-derived Pmin-containing OSP20 (5′-atcgctcgagGTCGACAGCGGAGACTCTAGAGGGTATATAATGGAAGCTCGACTTCCAGCTTGGCAATCCGGTACTGTTGGTAAAgaattcatcg-3′) and OSP21 (5′-cgatgaattc TTTACCAACAGTACCGGATTGCCAAGCTGGAAGTCGAGCTTCCATTATATACCCTCTAGAGTCTCCGCTGTCGACctcgagcgat-3′) were annealed, restricted with XhoI/EcoRI, and cloned into the corresponding sites (XhoI/EcoRI) of pSP15 | This work |
| pSP27 | Constitutive TSR expression vector (PhCMV-TSR-pAbGH). The Gal4 DNA-binding domain was PCR-amplified from pGal4 using OSP25 (5′-acgcgctagccaccATGAAGCTACTGTCTTCTATC-3′) and OSP26 (5′-acgcggatccCGATACAGTCAACTGTCTTTG-3′) and restricted with NheI/BamHI. The ligand-binding domain of hTRα was PCR-amplified from pTRα using OSP27 (5′-acgcggatccATGGACTTGG TTCTAGATGAC-3′) and OSP28 (5′-acgcgcggccgcttaGACTTCCTGATCCTCAAAGAC-3′) and restricted using BamHI/NotI. Both PCR products were ligated into pcDNA3.1(+) restricted with NheI/NotI. | This work |
| pSP28 | PUAS1-driven SEAP expression vector (PUAS1-SEAP-pASV40). PUAS1 was assembled by PCR-mediated amplification of minimal SV40 promoter from pSAM200 using OSP29 (5′-gagcgacgtccgga gtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgagatctcctaggaagcttTGCATCTCAATTAGTCAGCAACCATAG and OSP30 (5′- gagcgaattcAAGCTTTTTGCAAAAGCCTAGGCCTC-3′), restricted with AatII/EcoRI and cloned into the corresponding sites (AatII/EcoRI) of pWW124. | This work |
| pSP29 | PUAS2-driven SEAP expression vector (PUAS2-SEAP-pASV40). Oligonucleotides OSP31 (5′-gagcgacgtc CGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGagatctgagc-3′) and OSP32 (5′-gctcagatctCGGAGGA CAGTACTCCGCTCGGAGGACAGTACTCCGCTCGGAGGACAGTACTCCGCTCGGAGGACAGTACTCCGCTCGGAGGACAGTACTCCGgacgtcgctc-3′) were annealed, restricted with AatII/BglII, and cloned into the corresponding sites (AatII/BglII) of pSP28 | This work |
| pSP30 | PUAS5-driven SEAP expression vector (PUAS5-SEAP-pASV40). Oligonucleotides OSP33 (5′-gagcgacgtc CGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGcctgcagggagc-3′) and OSP34 (5′-gctccctgcaggCGG AGGACAGTACTCCGCTCGGAGGACAGTACTCCGCTCGGAGGACAGTACTCCGCTCGGAGGACAGTACTCCGCTCGGAGGACAGTACTCCGgacgtcgctc-3′) were annealed, restricted with AatII/SbfI, and ligated into the corresponding sites (AatII/SbfI) of pWW124 | This work |
| pSP31 | pUC57-derived vector containing hTSHAntag | This work |
| pSP32 | Constitutive hTSHAntag expression vector (PhCMV-hTSHAntag-pAbGH). hTSHAntag was excised from pSP31 with EcoRI/XbaI and cloned into the corresponding sites (EcoRI/XbaI) of pcDNA3.1(+) | This work |
| pSP34 | PUAS5-driven hTSHAntag expression vector (PUAS5-hTSHAntag-pASV40). hTSHAntag was excised from pSP31 using EcoRI/XbaI and cloned into the corresponding sites (EcoRI/XbaI) of pSP30 | This work |
| pSP35 | PUAS5-driven Citrine expression vector (PUAS5-Citrine-pASV40). Citrine was PCR-amplified from pDA134 using OSP35 (5′-acgcgaattcgccaccATGGTGAGCAAGGGCGAGGAGCTG-3′) and OSP36 (5′-acgcaagctt CTACTTGTACAGCTCGTCCATGCCCAGGGTG-3′), restricted with EcoRI/HindIII, and cloned into the corresponding sites EcoRI/HindIII of pDA134 | This work |
| pSP50 | PUAS2.1-driven SEAP expression vector (PUAS2.1-SEAP-pASV40). Oligonucleotides OSP61 (5′-gagcgacgtc CGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGagatctgagc-3′) and OSP62 (5′-gctcagatctCGGC GGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGgacgtcgctc-3′) were annealed, restricted with AatII/BglII, and cloned into the corresponding sites (AatII/BglII) of pSP29 | This work |
| pSP51 | PUAS5.1-driven SEAP expression vector (PUAS5.1-SEAP-pASV40). Oligonucleotides OSP63 (5′-gagcgacgtc CGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGcctgcagggagc-3′) and OSP64 (5′-gctccctgcaggCGGCG GTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGAGCGGCGGTCTTTCGTCCGgacgtcgctc-3′) were annealed, restricted with AatII/SbfI, and cloned into the corresponding sites (AatII/SbfI) of pWW124 | This work |
| pSP52 | PUAS3-driven SEAP expression vector (PUAS3-SEAP-pASV40). Oligonucleotides OSP65 (5′-gagcacggct CGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGctcgaggagc-3′) and OSP66 (5′-gctcctcgagCGGA GGACAGTACTCCGCTCGGAGGACAGTACTCCGCTCGGAGGACAGTACTCCGCTCGGAGGACAGTACTCCGCTCGGAGGACAGTACTCCGacggctgctc-3′) were annealed, restricted with MluI/XhoI, and ligated into the corresponding sites (MluI/XhoI) of pSP16 | This work |
Oligonucleotides: Restriction endonuclease-specific sites are underlined in oligonucleotide sequences. Annealing base pairs contained in oligonucleotide sequences are shown in capital letters. Citrine, improved version of YFP derived from Aequorea victoria; Gal4, Saccharomyces cerevisiae-derived Gal4 transcription factor; Gal4DBD, Gal4 transcription factor DNA binding domain; hCG, human CG hormone; hTRα, human thyroid hormone receptor-α; hTRαLBD, human thyroid hormone receptor-α ligand binding domain; hTSHR, human thyroid-stimulating hormone receptor; hTSHAntag, human stimulating hormone β- and α-chains linked by the hCG-derived C-terminal peptide containing mutations to eliminate N-linked glycosylation; Luc2, Photinus pyralis firefly luciferase; MCS, multiple cloning site; pAbGH, polyadenylation signal of the bovine growth hormone; pASV40, polyadenylation signal of the simian virus 40; PCREm, synthetic mammalian promoter containing a modified cAMP-response element (CREm-Pmin); PhCMV, human CMV immediate early promoter; PhCMVmin, minimal version of PhCMV; Pmin, pGL4.23-derived minimal promoter; PSPA, synthetic mammalian promoter containing Streptomyces pristinaespiralis butyrolactone-dependent transactivator binding site (OpapRI-PhCMVmin); PUAS1, synthetic mammalian promoter containing three Gal4-specific upstream activating sequence operator sites (UAS3-PSV40min); PUAS2, synthetic mammalian promoter containing five Gal4-specific upstream activating sequence operator sites (UAS5-PSV40min); PUAS2.1, synthetic mammalian promoter containing five modified Gal4-specific upstream activating sequence operator sites (UASm5-PSV40min); PUAS3, synthetic mammalian promoter containing five Gal4-specific upstream activating sequence operator sites (UAS5-Pmin); PUAS5, synthetic mammalian promoter containing five Gal4-specific upstream activating sequence operator sites (UAS5-PhCMVmin); PUAS5.1, synthetic mammalian promoter containing five modified Gal4-specific upstream activating sequence operator sites (UASm5-PhCMVmin); SPA, Streptomyces pristinaespiralis butyrolactone-dependent transactivator (SpbR-VP16); tTA, tetracycline-dependent transactivator; UAS, Saccharomyces cerevisiae-derived Gal4-specific upstream activator sequence; UASm, upstream activator sequence variant containing mutations.