LOCUS Exported 8927 bp ds-DNA circular SYN 30-SEP-2021 DEFINITION Expression of G protein-coupled receptors for PRESTO-Tango: parallel receptorome expression and screening via transcriptional output, with transcriptional activation following arrestin translocation. ACCESSION . VERSION . KEYWORDS TSHR-Tango SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8927) AUTHORS Kroeze WK, Sassano MF, Huang XP, Lansu K, McCorvy JD, Giguere PM, Sciaky N, Roth BL TITLE PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. JOURNAL Nat Struct Mol Biol. 2015 May;22(5):362-9. doi: 10.1038/nsmb.3014. Epub 2015 Apr 20. PUBMED 25895059 REFERENCE 2 (bases 1 to 8927) AUTHORS . TITLE Direct Submission JOURNAL Exported Sep 30, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8927 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 629..648 /label=T7 /note="T7 promoter, forward primer" promoter 629..647 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 3251..4252 /codon_start=1 /product="tetracycline-controlled transactivator, comprising a fusion of the tetracycline repressor TetR with the C-terminal activation domain of herpes simplex virus VP16" /label=tTA /note="In the Tet-Off(R) system, tTA binds to the Tet-responsive element and stimulates transcription only in the absence of tetracycline or doxycycline." /translation="RLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKN KRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPT EKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDS MPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEG LLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADA LDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG" primer_bind complement(3318..3337) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" primer_bind complement(4284..4301) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4290..4514 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 4560..4988 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4647..4666) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4857..4878 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4997..5017) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5002..5331 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5182..5317 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5244..5263 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 5398..6192 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(5452..5471) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 6062..6081 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 6366..6487 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6403..6422) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6457..6476 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(6536..6552) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6536..6552) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6549..6571) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6560..6576 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(6584..6614) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6629..6650 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6767..6784) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6938..7526) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7018..7037) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7697..8557) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 8320..8339 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8558..8662) /gene="bla" /label=AmpR promoter primer_bind complement(8737..8756) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg 661 gctagcgttt aaacttaagc ttggtaccga gctcggatcc actagtccag tgtggtggaa 721 ttctgcagat atccagcaca gtggcggccg cgccaccatg aagacgatca tcgccctgag 781 ctacatcttc tgcctggtat tcgccgacta caaggacgat gatgacgcca gcatcgatat 841 gcgccctgcc gatctgctgc agctggtgct tctccttgac cttccccgcg acttgggggg 901 catggggtgc agctcacccc cttgtgaatg tcatcaggag gaagactttc gagtgacctg 961 caaagacatt cagagaatcc catcactccc cccaagcaca cagactctga agctgatcga 1021 aacccatctg cggacaattc ctagccacgc cttctcaaat cttcccaaca tttctcgcat 1081 atacgtgtca atcgacgtaa cgcttcagca gttggaatct cattccttct ataatctgag 1141 caaggtgacc cacattgaaa ttcggaacac taggaacctc acgtacatag atcccgacgc 1201 acttaaggaa ctgccattgc tgaagttcct tggcattttc aatactggac tgaagatgtt 1261 tccagacctg accaaggtgt actccaccga catattcttt attttggaga tcaccgataa 1321 cccttatatg acaagtattc ctgttaacgc ttttcagggg ttgtgcaacg aaaccctgac 1381 gctcaagttg tacaataacg ggttcaccag cgtacagggt tacgccttca atgggaccaa 1441 gttggacgcc gtctacctca ataagaacaa gtatctcact gttattgata aggatgcatt 1501 cggcggggtg tactcaggcc cctccctcct ggatgttagc cagacaagtg tcaccgcatt 1561 gcccagtaag ggcctggagc acctgaaaga gctcatcgca cgcaatacat ggaccctgaa 1621 gaagctcccg ctttctttgt cctttctgca tctgaccagg gctgatctgt cttaccccag 1681 ccactgttgc gcctttaaaa atcagaagaa aattaggggc atcttggaga gtctgatgtg 1741 taatgaatct agcatgcaga gccttcggca gcggaaaagc gtgaacgctc tgaacagtcc 1801 tctccatcag gaatacgaag agaatttggg agactcaatc gtcgggtaca aggagaaatc 1861 caagttccag gatacccaca ataacgccca ctactatgtt ttttttgaag aacaggaaga 1921 cgagatcatc gggtttggcc aagagctgaa aaaccctcaa gaggagaccc tgcaggcctt 1981 cgatagccat tatgactata ctatctgtgg cgacagcgaa gacatggtgt gtactcctaa 2041 gagcgatgaa ttcaatccct gtgaggacat catgggctac aagtttctta ggatagtagt 2101 ttggtttgtt agtttgttgg ctttgcttgg aaacgtcttt gtgctcctga tccttctcac 2161 ttctcattac aaactgaatg ttcccaggtt tcttatgtgc aacctggcgt tcgccgattt 2221 ctgtatgggc atgtacttgc tgctcatcgc ttcagtcgat ctgtacacac attcagagta 2281 ctacaaccat gctatcgact ggcagaccgg accaggctgc aacacagccg gatttttcac 2341 cgtgttcgct tctgagctgt ccgtgtacac tctgactgtc attacattgg agcggtggta 2401 tgcaatcaca ttcgccatgc gactcgaccg gaagattcgg ctccgacacg cctgtgcaat 2461 aatggttggc gggtgggttt gctgtttcct gcttgctttg ctgcccctgg tcggtattag 2521 ctcctatgcg aaagtgtcca tatgcctgcc catggacaca gagactcccc tggccttggc 2581 ttatattgtg ttcgttctca ccctgaatat agtagccttc gtgatcgtct gttgctgtta 2641 cgtcaaaatt tatatcacag tgcgcaaccc acagtacaac ccgggggata aggatacaaa 2701 aattgccaag agaatggccg tgctcatttt cacggatttt atctgtatgg cacctatttc 2761 tttctacgcg ctctctgcca tcctgaacaa acctttgatc accgtttcca atagcaagat 2821 cctgctcgta ctcttctatc cactgaatag ctgtgccaac ccattccttt atgccatttt 2881 caccaaagct tttcagcgcg acgtgtttat tttgctttcc aaatttggaa tttgcaaacg 2941 acaagcccaa gcctatagag gccagcgggt gccccctaaa aattccaccg atattcaggt 3001 gcagaaggtt acgcatgaga tgcggcaagg cctccataat atggaggacg tgtacgagct 3061 gattgagaat agtcatctca caccaaaaaa acagggtcaa atcagtgagg agtacatgca 3121 aacagttctg atcgataccg gtggacgcac cccacccagc ctgggtcccc aagatgagtc 3181 ctgcaccacc gccagctcct ccctggccaa ggacacttca tcgaccggtg agaacctgta 3241 cttccagcta agattagata aaagtaaagt gattaacagc gcattagagc tgcttaatga 3301 ggtcggaatc gaaggtttaa caacccgtaa actcgcccag aagctaggtg tagagcagcc 3361 tacattgtat tggcatgtaa aaaataagcg ggctttgctc gacgccttag ccattgagat 3421 gttagatagg caccatactc acttttgccc tttagaaggg gaaagctggc aagatttttt 3481 acgtaataac gctaaaagtt ttagatgtgc tttactaagt catcgcgatg gagcaaaagt 3541 acatttaggt acacggccta cagaaaaaca gtatgaaact ctcgaaaatc aattagcctt 3601 tttatgccaa caaggttttt cactagagaa tgcattatat gcactcagcg ctgtggggca 3661 ttttacttta ggttgcgtat tggaagatca agagcatcaa gtcgctaaag aagaaaggga 3721 aacacctact actgatagta tgccgccatt attacgacaa gctatcgaat tatttgatca 3781 ccaaggtgca gagccagcct tcttattcgg ccttgaattg atcatatgcg gattagaaaa 3841 acaacttaaa tgtgaaagtg ggtccgcgta cagccgcgcg cgtacgaaaa acaattacgg 3901 gtctaccatc gagggcctgc tcgatctccc ggacgacgac gcccccgaag aggcggggct 3961 ggcggctccg cgcctgtcct ttctccccgc gggacacacg cgcagactgt cgacggcccc 4021 cccgaccgat gtcagcctgg gggacgagct ccacttagac ggcgaggacg tggcgatggc 4081 gcatgccgac gcgctagacg atttcgatct ggacatgttg ggggacgggg attccccggg 4141 tccgggattt accccccacg actccgcccc ctacggcgct ctggatatgg ccgacttcga 4201 gtttgagcag atgtttaccg atgcccttgg aattgacgag tacggtgggt agtctagagg 4261 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt 4321 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta 4381 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg 4441 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc 4501 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca 4561 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 4621 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 4681 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 4741 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc 4801 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 4861 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata 4921 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 4981 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca 5041 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc 5101 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 5161 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 5221 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag 5281 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg 5341 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg 5401 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 5461 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 5521 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 5581 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 5641 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 5701 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 5761 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 5821 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 5881 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc 5941 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 6001 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 6061 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 6121 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 6181 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 6241 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 6301 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 6361 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 6421 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 6481 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat 6541 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 6601 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 6661 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 6721 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 6781 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 6841 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 6901 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 6961 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 7021 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 7081 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 7141 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 7201 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 7261 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 7321 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 7381 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 7441 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 7501 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 7561 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 7621 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 7681 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 7741 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 7801 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 7861 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 7921 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 7981 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 8041 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 8101 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 8161 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 8221 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 8281 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 8341 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 8401 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 8461 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 8521 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 8581 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 8641 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat 8701 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata 8761 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa 8821 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt 8881 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgtt //