LOCUS Exported 7709 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 PTGER2-Tango SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7709) 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 7709) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7709 /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 2033..3034 /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(2100..2119) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" primer_bind complement(3066..3083) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3072..3296 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3342..3770 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3429..3448) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3639..3660 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3779..3799) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3784..4113 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3964..4099 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4026..4045 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4180..4974 /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(4234..4253) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4844..4863 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5148..5269 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5185..5204) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5239..5258 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5318..5334) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5318..5334) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5331..5353) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5342..5358 /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(5366..5396) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5411..5432 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5549..5566) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5720..6308) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5800..5819) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6479..7339) /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 7102..7121 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7340..7444) /gene="bla" /label=AmpR promoter primer_bind complement(7519..7538) /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 gggaaacgct tctaatgatt cccaaagcga agactgtgag accaggcagt ggctcccccc 901 cggtgaatct ccagcgattt cctccgtcat gttctctgcc ggtgttctcg gtaacttgat 961 cgcgcttgct ctcctcgcta ggcgctggag aggcgacgtg gggtgcagcg cagggagacg 1021 ctcatcactg agtctgtttc acgtgctggt cactgaattg gtcttcaccg atctcttggg 1081 cacatgtctg atctcccccg tggtgctggc tagctatgcc cgcaatcaga cattggtcgc 1141 cttggcccca gagagccgcg cttgtaccta tttcgctttc gccatgacat ttttttctct 1201 cgcaacaatg ctcatgctgt ttgccatggc tttggagcgg tacctgagta tcggccaccc 1261 atatttctac cagcggcggg tcagcaggtc cgggggactg gcagtgctcc cagtgattta 1321 cgccgtgtca ctgttgtttt gttcactgcc gctgctggac tatggtcagt atgtgcaata 1381 ctgtcccgga acatggtgtt ttattcgaca tgggaggaca gcctacctcc aactctacgc 1441 tacactgttg ctgctcctca tcgtgtccgt cctggcctgc aatttctctg taattctcaa 1501 cctcattcgc atgcatcgga gaagccgccg gagcaggtgt ggcccaagcc tgggatctgg 1561 acgcggtggg ccaggtgcaa ggcgccgcgg agaaagggtt agcatggcag aagaaacgga 1621 ccacctgatt ctcctggcga taatgaccat cacttttgcc gtgtgcagcc ttccttttac 1681 aatcttcgca tacatgaacg agaccagtag tagaaaagaa aagtgggacc tgcaggcttt 1741 gcgatttctc tctatcaact ctatcattga cccctgggtt tttgccattc tgcgccctcc 1801 agtcctgagg ctgatgcgct ccgtcttgtg ttgtagaata agcctgagga cccaggacgc 1861 cacccagaca agttgcagta cacagagcga tgcttccaag caagctgatc tcatcgatac 1921 cggtggacgc accccaccca gcctgggtcc ccaagatgag tcctgcacca ccgccagctc 1981 ctccctggcc aaggacactt catcgaccgg tgagaacctg tacttccagc taagattaga 2041 taaaagtaaa gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt 2101 aacaacccgt aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt 2161 aaaaaataag cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac 2221 tcacttttgc cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag 2281 ttttagatgt gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc 2341 tacagaaaaa cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt 2401 ttcactagag aatgcattat atgcactcag cgctgtgggg cattttactt taggttgcgt 2461 attggaagat caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag 2521 tatgccgcca ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc 2581 cttcttattc ggccttgaat tgatcatatg cggattagaa aaacaactta aatgtgaaag 2641 tgggtccgcg tacagccgcg cgcgtacgaa aaacaattac gggtctacca tcgagggcct 2701 gctcgatctc ccggacgacg acgcccccga agaggcgggg ctggcggctc cgcgcctgtc 2761 ctttctcccc gcgggacaca cgcgcagact gtcgacggcc cccccgaccg atgtcagcct 2821 gggggacgag ctccacttag acggcgagga cgtggcgatg gcgcatgccg acgcgctaga 2881 cgatttcgat ctggacatgt tgggggacgg ggattccccg ggtccgggat ttacccccca 2941 cgactccgcc ccctacggcg ctctggatat ggccgacttc gagtttgagc agatgtttac 3001 cgatgccctt ggaattgacg agtacggtgg gtagtctaga gggcccgttt aaacccgctg 3061 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 3121 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 3181 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 3241 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc 3301 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc 3361 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 3421 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 3481 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 3541 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 3601 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 3661 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 3721 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg 3781 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 3841 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 3901 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 3961 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 4021 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga 4081 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt 4141 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg 4201 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 4261 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 4321 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta 4381 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 4441 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 4501 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 4561 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 4621 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 4681 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc 4741 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 4801 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 4861 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 4921 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga 4981 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt 5041 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga 5101 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg 5161 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 5221 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta 5281 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 5341 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 5401 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 5461 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 5521 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 5581 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 5641 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 5701 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 5761 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 5821 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 5881 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 5941 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 6001 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 6061 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 6121 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 6181 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 6241 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 6301 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 6361 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 6421 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 6481 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 6541 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 6601 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 6661 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 6721 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 6781 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 6841 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 6901 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 6961 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 7021 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 7081 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 7141 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 7201 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 7261 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 7321 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 7381 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 7441 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc 7501 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct 7561 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag 7621 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct 7681 tcgcgatgta cgggccagat atacgcgtt //