LOCUS Exported 8705 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 ELTD1-Tango SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8705) 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 8705) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8705 /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 3029..4030 /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(3096..3115) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" primer_bind complement(4062..4079) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4068..4292 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 4338..4766 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4425..4444) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4635..4656 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4775..4795) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4780..5109 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4960..5095 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5022..5041 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 5176..5970 /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(5230..5249) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5840..5859 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 6144..6265 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6181..6200) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6235..6254 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(6314..6330) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6314..6330) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6327..6349) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6338..6354 /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(6362..6392) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6407..6428 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6545..6562) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6716..7304) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6796..6815) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7475..8335) /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 8098..8117 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8336..8440) /gene="bla" /label=AmpR promoter primer_bind complement(8515..8534) /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 gaagagactg ccactgttgg tggtgttttc tactctcctg aattgtagtt atacccagaa 901 ctgcaccaag acaccatgtc ttcctaacgc aaaatgtgaa atccggaatg gtattgaagc 961 ctgttactgt aacatgggtt tttctggcaa cggggtgacc atatgtgagg acgataacga 1021 gtgcggcaat ttgacacaga gttgtggaga aaatgccaac tgtaccaata cagaggggtc 1081 ctattactgc atgtgcgtcc cgggctttcg gtcctcatcc aatcaagaca gatttattac 1141 gaacgacggg accgtgtgca tcgagaacgt taacgcaaac tgccacctgg acaatgtgtg 1201 cattgcagct aacattaata agacactgac caaaatccgc tcaattaagg aacccgtggc 1261 cctgctccaa gaggtgtaca gaaacagtgt caccgatctg tcccccacag atatcattac 1321 ctatatcgag attctggccg agagcagctc tctgttggga tataaaaaca acacgatctc 1381 cgctaaggat acccttagca attctactct gaccgaattc gtgaaaactg ttaacaactt 1441 tgttcagcga gacacattcg tggtgtggga taagctgagt gtcaatcaca ggagaaccca 1501 cctcacaaag ctcatgcata ccgtggagca ggctacactt agaattagcc agagtttcca 1561 gaaaaccaca gagttcgata cgaactctac agatatcgcc cttaaagtat tcttctttga 1621 ttcctataac atgaagcata tacaccctca tatgaatatg gacggcgact acatcaacat 1681 cttccctaaa cggaaggcgg cctacgattc taacggcaat gtggccgtcg ccttcgtgta 1741 ctacaagtcc atcggccctt tgctgtcttc ctcagataat ttcctgctca aaccccagaa 1801 ctatgacaat tcagaagagg aagagcgcgt gattagctcc gttatcagtg ttagtatgtc 1861 ctcaaacccc ccaacactgt atgagttgga aaagatcaca ttcactctgt ctcatcggaa 1921 ggtgaccgat agatacagga gcctctgtgc tttttggaat tacagcccag acacaatgaa 1981 tggatcttgg agtagtgagg gatgtgagct cacctacagt aatgagaccc acaccagctg 2041 caggtgtaac catctgacac acttcgctat tctcatgtcc tcaggcccgt ccatcgggat 2101 caaggactac aacatcctta ccaggattac gcaactcggc ataattatta gcctgatttg 2161 tctggctatt tgcattttta ctttttggtt tttcagcgag attcaatcta ccaggacaac 2221 cattcataag aatttgtgct gtagcctctt cctcgccgag ttggtctttt tggtcggtat 2281 caacactaat actaacaaac ttttttgttc aattattgcc ggcttgctcc attacttctt 2341 cctcgcagca ttcgcgtgga tgtgtataga gggtatccac ctttatctca ttgtcgtcgg 2401 cgtgatatat aataagggct ttctgcataa gaacttctat atatttggat atctctcccc 2461 agcagtagtt gttggtttta gtgccgcatt gggctacagg tactatggaa cgaccaaggt 2521 gtgttggctg tcaactgaaa acaatttcat ctggtcattt atcggcccag cgtgcctgat 2581 catcctggtc aacttgctgg cattcggcgt cattatctac aaggtgttta ggcacacggc 2641 cgggctgaag cctgaagtct catgcttcga gaacattcga tcttgcgctc ggggcgcact 2701 cgcgcttctc ttccttcttg gtaccacatg gatctttgga gtgctgcatg tcgtgcacgc 2761 atctgtggtg accgcttacc ttttcacagt ttcaaacgcc ttccaaggaa tgttcatttt 2821 cttgttcctc tgcgtcttga gtcggaaaat tcaggaagag tactatcgcc tgttcaaaaa 2881 cgtaccttgc tgcttcggct gccttaggat cgataccggt ggacgcaccc cacccagcct 2941 gggtccccaa gatgagtcct gcaccaccgc cagctcctcc ctggccaagg acacttcatc 3001 gaccggtgag aacctgtact tccagctaag attagataaa agtaaagtga ttaacagcgc 3061 attagagctg cttaatgagg tcggaatcga aggtttaaca acccgtaaac tcgcccagaa 3121 gctaggtgta gagcagccta cattgtattg gcatgtaaaa aataagcggg ctttgctcga 3181 cgccttagcc attgagatgt tagataggca ccatactcac ttttgccctt tagaagggga 3241 aagctggcaa gattttttac gtaataacgc taaaagtttt agatgtgctt tactaagtca 3301 tcgcgatgga gcaaaagtac atttaggtac acggcctaca gaaaaacagt atgaaactct 3361 cgaaaatcaa ttagcctttt tatgccaaca aggtttttca ctagagaatg cattatatgc 3421 actcagcgct gtggggcatt ttactttagg ttgcgtattg gaagatcaag agcatcaagt 3481 cgctaaagaa gaaagggaaa cacctactac tgatagtatg ccgccattat tacgacaagc 3541 tatcgaatta tttgatcacc aaggtgcaga gccagccttc ttattcggcc ttgaattgat 3601 catatgcgga ttagaaaaac aacttaaatg tgaaagtggg tccgcgtaca gccgcgcgcg 3661 tacgaaaaac aattacgggt ctaccatcga gggcctgctc gatctcccgg acgacgacgc 3721 ccccgaagag gcggggctgg cggctccgcg cctgtccttt ctccccgcgg gacacacgcg 3781 cagactgtcg acggcccccc cgaccgatgt cagcctgggg gacgagctcc acttagacgg 3841 cgaggacgtg gcgatggcgc atgccgacgc gctagacgat ttcgatctgg acatgttggg 3901 ggacggggat tccccgggtc cgggatttac cccccacgac tccgccccct acggcgctct 3961 ggatatggcc gacttcgagt ttgagcagat gtttaccgat gcccttggaa ttgacgagta 4021 cggtgggtag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag 4081 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 4141 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 4201 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 4261 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg 4321 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 4381 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 4441 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 4501 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 4561 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 4621 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 4681 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 4741 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga 4801 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 4861 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 4921 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc 4981 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag 5041 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc 5101 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga 5161 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg 5221 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc 5281 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt 5341 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt 5401 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc 5461 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc 5521 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac 5581 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag 5641 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag 5701 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat 5761 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg 5821 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa 5881 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc 5941 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc 6001 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt 6061 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca 6121 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa 6181 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 6241 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct 6301 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 6361 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 6421 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 6481 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 6541 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 6601 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 6661 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 6721 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 6781 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 6841 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 6901 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 6961 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 7021 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 7081 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 7141 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 7201 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 7261 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 7321 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 7381 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 7441 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 7501 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 7561 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 7621 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 7681 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 7741 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 7801 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 7861 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 7921 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 7981 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 8041 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 8101 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 8161 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 8221 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 8281 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 8341 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 8401 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 8461 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat 8521 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc 8581 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat 8641 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac 8701 gcgtt //