LOCUS Exported 7796 bp ds-DNA circular SYN 12-MAY-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 HCA3-Tango SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7796) 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 7796) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7796 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 2354..3355 /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(2421..2440) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" primer_bind complement(3387..3404) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3393..3617 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3663..4091 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3750..3769) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3960..3981 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4100..4120) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4105..4434 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4285..4420 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4347..4366 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4501..5295 /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(4555..4574) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5165..5184 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5469..5590 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5506..5525) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5560..5579 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5639..5655) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5639..5655) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5652..5674) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5663..5679 /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(5687..5717) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5732..5753 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5870..5887) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6041..6629) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6121..6140) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6800..7660) /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 7423..7442 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7661..7765) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc 961 agatatccag cacagtggcg gccgcgccac catgaagacg atcatcgccc tgagctacat 1021 cttctgcctg gtattcgccg actacaagga cgatgatgac gccagcatcg atatgaatcg 1081 ccaccacctg caggatcact ttctcgaaat cgacaagaag aactgttgcg tcttccggga 1141 cgattttata gccaaagtgc tcccacccgt gctgggcctg gagttcatct ttggcctctt 1201 gggcaacggg ttggcccttt ggatcttctg cttccatctc aagagctgga aatcttcccg 1261 gattttcctc ttcaatttgg ccgtagcaga tttcctgctc atcatctgtc ttcctttcgt 1321 catggactac tatgtgcgca gaagcgattg gaagtttggt gacattccct gccgcttggt 1381 tttgtttatg tttgctatga accgacaggg tagcatcatc tttctgaccg tcgttgccgt 1441 cgaccgatat tttcgggtgg tgcatcctca ccatgccttg aacaaaatat ccaactggac 1501 agccgctata atttcttgct tgctgtgggg cattactgtg gggctgactg tgcaccttct 1561 gaagaagaaa ttgctgatcc agaatgggac cgcgaacgta tgcatttctt tctctatatg 1621 tcatacattt cgatggcatg aggccatgtt cctgctggag ttttttcttc cactcgggat 1681 tatccttttc tgtagtgcta ggatcatctg gagtttgcgg caacgccaaa tggatcggca 1741 tgctaaaatt aagcgagcca taacgttcat tatggtggtt gccatagtgt tcgtgatctg 1801 ttttctgcct agcgtcgtcg tgaggatcca catcttttgg ctgctgcaca cctctgggac 1861 ccagaactgc gaagtgtacc gatcagtaga tctcgcgttt tttatcacat tgtcctttac 1921 ttacatgaac tccatgctcg accctgtggt ttactacttc agttctccct cttttccaaa 1981 cttcttttcc accctgatta atcggtgcct tcaaaggaag attacaggag agcctgataa 2041 taacaggagc acttcagtcg agctgaccgg agatcccaac aagactcggg gagcccctga 2101 ggcacttatc gccaattctg gagaaccctg gagccctagc tacctggggc caacatctaa 2161 taaccattct aagaaggggc actgccacca ggagccagcc agcttggaga aacagttggg 2221 ctgttgtatc gaaatcgata ccggtggacg caccccaccc agcctgggtc cccaagatga 2281 gtcctgcacc accgccagct cctccctggc caaggacact tcatcgaccg gtgagaacct 2341 gtacttccag ctaagattag ataaaagtaa agtgattaac agcgcattag agctgcttaa 2401 tgaggtcgga atcgaaggtt taacaacccg taaactcgcc cagaagctag gtgtagagca 2461 gcctacattg tattggcatg taaaaaataa gcgggctttg ctcgacgcct tagccattga 2521 gatgttagat aggcaccata ctcacttttg ccctttagaa ggggaaagct ggcaagattt 2581 tttacgtaat aacgctaaaa gttttagatg tgctttacta agtcatcgcg atggagcaaa 2641 agtacattta ggtacacggc ctacagaaaa acagtatgaa actctcgaaa atcaattagc 2701 ctttttatgc caacaaggtt tttcactaga gaatgcatta tatgcactca gcgctgtggg 2761 gcattttact ttaggttgcg tattggaaga tcaagagcat caagtcgcta aagaagaaag 2821 ggaaacacct actactgata gtatgccgcc attattacga caagctatcg aattatttga 2881 tcaccaaggt gcagagccag ccttcttatt cggccttgaa ttgatcatat gcggattaga 2941 aaaacaactt aaatgtgaaa gtgggtccgc gtacagccgc gcgcgtacga aaaacaatta 3001 cgggtctacc atcgagggcc tgctcgatct cccggacgac gacgcccccg aagaggcggg 3061 gctggcggct ccgcgcctgt cctttctccc cgcgggacac acgcgcagac tgtcgacggc 3121 ccccccgacc gatgtcagcc tgggggacga gctccactta gacggcgagg acgtggcgat 3181 ggcgcatgcc gacgcgctag acgatttcga tctggacatg ttgggggacg gggattcccc 3241 gggtccggga tttacccccc acgactccgc cccctacggc gctctggata tggccgactt 3301 cgagtttgag cagatgttta ccgatgccct tggaattgac gagtacggtg ggtagtctag 3361 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 3421 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 3481 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 3541 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 3601 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc 3661 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 3721 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 3781 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 3841 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 3901 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 3961 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 4021 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 4081 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc 4141 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag 4201 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 4261 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 4321 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct 4381 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc 4441 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc 4501 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 4561 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 4621 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 4681 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 4741 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 4801 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 4861 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 4921 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 4981 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac 5041 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 5101 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 5161 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 5221 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 5281 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc 5341 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg 5401 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg 5461 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 5521 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 5581 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt 5641 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg 5701 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt 5761 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 5821 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 5881 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 5941 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 6001 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 6061 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 6121 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 6181 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 6241 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 6301 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 6361 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 6421 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 6481 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 6541 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6601 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 6661 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 6721 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 6781 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 6841 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 6901 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 6961 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 7021 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 7081 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 7141 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 7201 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 7261 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 7321 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 7381 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 7441 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 7501 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 7561 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 7621 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 7681 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 7741 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc //