LOCUS       sequence-485939-        7589 bp    DNA     circular SYN 15-SEP-2025
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    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7589)
  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 7589)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nsmb.3014";
            journalName: "Nat Struct Mol Biol"; date: "2015-05"; volume: "22"; 
            issue: "5"; pages: "362-9"
COMMENT     Sequence Label: ADORA3-Tango
FEATURES             Location/Qualifiers
     source          1..7589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(844..863)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(845..863)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(908..1111)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(937..957)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1112..1491
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1663..1682
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1757..1861
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1862..1930,1931..2722)
                     /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     complement(2080..2099)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2893..3481
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3382..3401
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3635..3652
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3769..3790
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3805..3810,3811..3828,3829..3835)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3843..3859
                     /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)."
     primer_bind     3848..3870
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3867..3883
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3867..3883
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3932..4053
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3943..3962)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3997..4016
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4227..5021)
                     /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(4338..4357)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4948..4967
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5088..5417)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5102..5237
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5156..5175)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5402..5422
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5431..5859)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5541..5562)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5753..5772
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5905..6129
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6118..6135
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(6167..6553,6554..7168))
                     /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     7082..7101
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
ORIGIN
        1 gtttccttgc tgtttgacag attgagactg agcttgttcc ttatgatata aaagatatcc
       61 aggtatatag cacacatgac caccagcggg ataaatatcc aagtcagaaa gctgaagtaa
      121 accatataat ccattcgcat cacggataca aactgacaac tcaggaaagt aacgttccgg
      181 tggtattcag atgtcagctt catgttccac ccaaacatag gggtcaggcc caccaagaag
      241 ctaacaagcc aacacaggcc cagagccagc catattctgc ggtgggtggt aacccgctta
      301 taccgcacag tcaacttgac gcgcaggtac cgatcaacag cgatagccag aaggctcata
      361 atgctggcgt gcgtgaagat cagcagcaga caagtcatga acaagcagga gtaaaaatgg
      421 attgtgatcc cgagactgac cactatggcc agaggcatta ccaacacacc cacagcaatg
      481 tcggccagtg ccagtgacac aataaaataa aatgttgtag tctggagact tggattcaac
      541 ttgaccacac atataaccag gacattcccc acaatagcgc acagaccaat gaatatctcc
      601 atagtgatgt atgtaacgtt agccagagac aaggcggtag agttgtttgg catatcgatg
      661 ctggcgtcat catcgtcctt gtagtcggcg aataccaggc agaagatgta gctcagggcg
      721 atgatcgtct tcatggtggc gcggccgcca ctgtgctgga tatctgcaga attccaccac
      781 actggactag tggatccgag ctcggtacca agcttaagtt taaacgctag ccagcttggg
      841 tctccctata gtgagtcgta ttaatttcga taagccagta agcagtgggt tctctagtta
      901 gccagagagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
      961 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     1021 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     1081 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     1141 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc
     1201 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     1261 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     1321 gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc
     1381 agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa
     1441 ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt caacgcgtat
     1501 atctggcccg tacatcgcga agcagcgcaa aacgcctaac cctaagcaga ttcttcatgc
     1561 aattgtcggt caagccttgc cttgttgtag cttaaatttt gctcgcgcac tactcagcga
     1621 cctccaacac acaagcaggg agcagatact ggcttaacta tgcggcatca gagcagattg
     1681 tactgagagt gcaccatagg ggatcgggag atctcccgat ccgtcgacgt caggtggcac
     1741 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1801 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     1861 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     1921 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     1981 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2041 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2101 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2161 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2221 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2281 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     2341 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     2401 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     2461 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     2521 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     2581 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     2641 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     2701 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     2761 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     2821 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     2881 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2941 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3001 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     3061 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3121 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3181 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3241 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3301 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3361 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3421 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3481 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3541 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3601 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3661 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3721 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     3781 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     3841 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     3901 ctagctagag gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc
     3961 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4021 tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag
     4081 atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct
     4141 ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc
     4201 ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
     4261 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     4321 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     4381 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     4441 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     4501 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     4561 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     4621 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     4681 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     4741 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     4801 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     4861 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     4921 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     4981 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     5041 gtctcttgat cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct
     5101 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg
     5161 gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
     5221 ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga
     5281 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct
     5341 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     5401 caccctaact gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct
     5461 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     5521 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     5581 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     5641 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     5701 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     5761 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     5821 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc
     5881 agctggttct ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat
     5941 tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac
     6001 acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg
     6061 caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag
     6121 aaggcacagt cgaggctgat cagcgggttt aaacgggccc tctagactac ccaccgtact
     6181 cgtcaattcc aagggcatcg gtaaacatct gctcaaactc gaagtcggcc atatccagag
     6241 cgccgtaggg ggcggagtcg tggggggtaa atcccggacc cggggaatcc ccgtccccca
     6301 acatgtccag atcgaaatcg tctagcgcgt cggcatgcgc catcgccacg tcctcgccgt
     6361 ctaagtggag ctcgtccccc aggctgacat cggtcggggg ggccgtcgac agtctgcgcg
     6421 tgtgtcccgc ggggagaaag gacaggcgcg gagccgccag ccccgcctct tcgggggcgt
     6481 cgtcgtccgg gagatcgagc aggccctcga tggtagaccc gtaattgttt ttcgtacgcg
     6541 cgcggctgta cgcggaccca ctttcacatt taagttgttt ttctaatccg catatgatca
     6601 attcaaggcc gaataagaag gctggctctg caccttggtg atcaaataat tcgatagctt
     6661 gtcgtaataa tggcggcata ctatcagtag taggtgtttc cctttcttct ttagcgactt
     6721 gatgctcttg atcttccaat acgcaaccta aagtaaaatg ccccacagcg ctgagtgcat
     6781 ataatgcatt ctctagtgaa aaaccttgtt ggcataaaaa ggctaattga ttttcgagag
     6841 tttcatactg tttttctgta ggccgtgtac ctaaatgtac ttttgctcca tcgcgatgac
     6901 ttagtaaagc acatctaaaa cttttagcgt tattacgtaa aaaatcttgc cagctttccc
     6961 cttctaaagg gcaaaagtga gtatggtgcc tatctaacat ctcaatggct aaggcgtcga
     7021 gcaaagcccg cttatttttt acatgccaat acaatgtagg ctgctctaca cctagcttct
     7081 gggcgagttt acgggttgtt aaaccttcga ttccgacctc attaagcagc tctaatgcgc
     7141 tgttaatcac tttactttta tctaatctta gctggaagta caggttctca ccggtcgatg
     7201 aagtgtcctt ggccagggag gagctggcgg tggtgcagga ctcatcttgg ggacccaggc
     7261 tgggtggggt gcgtccaccg gtatcgatct ctgaattctt ttcgatgctc gtatccaggc
     7321 tatcagatgg gtggcatacg acacaggctt tcagaatcag caggtaggtt tccttaaact
     7381 ttttaatctt gtaagcatac actatgggat tcatcatact atttgcgtgg ctcaaaagta
     7441 ttcccatgta gagtaccagc tgcggcacct ctccgttaaa ataaatgata cagttgataa
     7501 tggacagtgg cagccaactc agagcgaaaa gaaagagcac caggaagagg ctcttagcag
     7561 tcttgaattc gcgcccataa aaggcacca
//
