LOCUS       sequence-417858-        7751 bp    DNA     circular SYN 15-DEC-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 7751)
  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 7751)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 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: OPRD1-Tango
FEATURES             Location/Qualifiers
     source          1..7751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             join(2075..2689,2690..3076)
                     /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(2142..2161)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3108..3125)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3114..3338
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3384..3812
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3471..3490)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3681..3702
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3821..3841)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3826..4155
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4006..4141
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4068..4087
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4222..5016
                     /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(4276..4295)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4886..4905
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5190..5311
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5227..5246)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5281..5300
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5360..5376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5360..5376)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5373..5395)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5384..5400
                     /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(join(5408..5414,5415..5432,5433..5438))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5453..5474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5591..5608)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5762..6350)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5842..5861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6521..7312,7313..7381))
                     /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     7144..7163
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7382..7486)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7561..7580)
                     /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 ggaaccagcc cctagtgcag gcgcagaatt gcagccccct ctgtttgcca acgcaagtga
      901 cgcctaccct tccgcgtgcc ccagcgcagg ggctaacgcc agcggccctc cgggggcaag
      961 aagcgctagc agtctggccc tggctattgc cattaccgcc ctgtattccg ccgtgtgtgc
     1021 agtgggactt ctgggtaacg tgctggtgat gttcgggatt gtgcgctaca ccaagatgaa
     1081 aaccgcaacc aacatttaca tttttaacct ggctttggcc gacgcactgg ccaccagtac
     1141 cctgccattc caaagcgcca aatacctgat ggaaacgtgg ccgtttgggg agttgctgtg
     1201 taaggccgtg ctgagcattg attactacaa catgtttacc tccatcttta ctctgacaat
     1261 gatgagtgtg gatcggtata ttgcagtctg ccacccagtc aaggccctgg atttccggac
     1321 tcccgcaaag gccaaactga taaatatatg tatctgggtc ctggcctcag gcgtgggagt
     1381 acccattatg gtgatggctg tgacacggcc ccgggatggg gccgttgtct gcatgcttca
     1441 atttcccagc ccctcctggt actgggatac cgtcactaag atctgtgtct tcctgtttgc
     1501 gtttgtggta ccaatcctga tcataacggt ctgctacggg ctgatgttgc tgaggctgag
     1561 gagtgtgagg ctcctgagtg ggagtaaaga aaaagatcga agtctgcgcc ggattacacg
     1621 gatggttctg gtagtggtgg gcgcgttcgt ggtgtgttgg gcccccatcc acatattcgt
     1681 gattgtgtgg acattggtcg acattgaccg ccgagatcct ctggtggtag ctgctctcca
     1741 tctgtgcatc gccttgggct atgccaatag ttctctgaat ccggtgctgt atgccttcct
     1801 ggacgagaac ttcaagagat gttttcggca gctgtgtagg aagccttgtg ggcgacccga
     1861 cccttcaagc ttcagccgag ctcgcgaagc aacagcaaga gagagggtaa ccgcctgtac
     1921 tcccagcgat gggcccgggg gtggagccgc cgcgatcgat accggtggac gcaccccacc
     1981 cagcctgggt ccccaagatg agtcctgcac caccgccagc tcctccctgg ccaaggacac
     2041 ttcatcgacc ggtgagaacc tgtacttcca gctaagatta gataaaagta aagtgattaa
     2101 cagcgcatta gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc gtaaactcgc
     2161 ccagaagcta ggtgtagagc agcctacatt gtattggcat gtaaaaaata agcgggcttt
     2221 gctcgacgcc ttagccattg agatgttaga taggcaccat actcactttt gccctttaga
     2281 aggggaaagc tggcaagatt ttttacgtaa taacgctaaa agttttagat gtgctttact
     2341 aagtcatcgc gatggagcaa aagtacattt aggtacacgg cctacagaaa aacagtatga
     2401 aactctcgaa aatcaattag cctttttatg ccaacaaggt ttttcactag agaatgcatt
     2461 atatgcactc agcgctgtgg ggcattttac tttaggttgc gtattggaag atcaagagca
     2521 tcaagtcgct aaagaagaaa gggaaacacc tactactgat agtatgccgc cattattacg
     2581 acaagctatc gaattatttg atcaccaagg tgcagagcca gccttcttat tcggccttga
     2641 attgatcata tgcggattag aaaaacaact taaatgtgaa agtgggtccg cgtacagccg
     2701 cgcgcgtacg aaaaacaatt acgggtctac catcgagggc ctgctcgatc tcccggacga
     2761 cgacgccccc gaagaggcgg ggctggcggc tccgcgcctg tcctttctcc ccgcgggaca
     2821 cacgcgcaga ctgtcgacgg cccccccgac cgatgtcagc ctgggggacg agctccactt
     2881 agacggcgag gacgtggcga tggcgcatgc cgacgcgcta gacgatttcg atctggacat
     2941 gttgggggac ggggattccc cgggtccggg atttaccccc cacgactccg ccccctacgg
     3001 cgctctggat atggccgact tcgagtttga gcagatgttt accgatgccc ttggaattga
     3061 cgagtacggt gggtagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     3121 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3181 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3241 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3301 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3361 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3421 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3481 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3541 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3601 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3661 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3721 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3781 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     3841 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3901 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3961 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4021 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4081 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4141 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     4201 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4261 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4321 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4381 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     4441 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4501 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4561 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4621 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4681 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4741 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4801 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4861 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4921 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4981 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5041 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     5101 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5161 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     5221 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5281 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5341 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5401 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5461 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5521 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5581 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5641 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5701 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5761 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5821 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5881 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5941 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6001 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6061 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6121 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6181 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6241 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6301 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6361 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6421 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6481 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6541 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6601 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6661 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6721 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6781 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6841 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6901 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6961 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7021 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7081 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7141 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7201 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7261 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7321 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7381 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7441 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7501 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     7561 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     7621 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     7681 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     7741 atatacgcgt t
//
