LOCUS       sequence-456527-        7712 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 7712)
  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 7712)
  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: HRH2-Tango
FEATURES             Location/Qualifiers
     source          1..7712
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             join(2270..2884,2885..3271)
                     /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(2337..2356)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3303..3320)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3309..3533
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3579..4007
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3666..3685)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3876..3897
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4016..4036)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4021..4350
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4201..4336
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4263..4282
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4417..5211
                     /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(4471..4490)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5081..5100
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5385..5506
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5422..5441)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5476..5495
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5555..5571)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5555..5571)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5568..5590)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5579..5595
                     /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(5603..5609,5610..5627,5628..5633))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5648..5669
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5786..5803)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5957..6545)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6037..6056)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6716..7507,7508..7576))
                     /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     7339..7358
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7577..7681)
                     /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 atatggctcc
     1081 taatgggact gcctcctcct tctgccttga ctctactgcc tgcaaaataa ctattacagt
     1141 ggtactcgcc gtcctgatac tgattactgt tgcgggtaat gtcgtagtgt gtctggccgt
     1201 cggccttaac cggagactga gaaaccttac taactgcttc attgtgagcc tggcgatcac
     1261 tgatctcctc ctgggcctcc tggtgttgcc ctttagtgca atttaccagc tgtcatgtaa
     1321 gtggtccttc ggaaaagtat tctgcaatat ttatacgagc ctggatgtga tgctgtgcac
     1381 ggcctccata ctgaatctgt tcatgatttc ccttgatcgg tattgcgccg tgatggatcc
     1441 cctgcggtac ccagtcttgg tgacccccgt tagagtggcg attagtctgg tcctcatttg
     1501 ggttatcagc attaccctct ccttcctgag catccatctg ggctggaact ccagaaacga
     1561 gacctctaag gggaaccaca caaccagtaa gtgcaaggtg caggttaacg aggtttacgg
     1621 gctggtagat ggccttgtga ctttttactt gcccctcctg attatgtgta taacttatta
     1681 ccggattttc aaggtggcca gagaccaggc caaacgcata aaccacatca gctcctggaa
     1741 agccgcaacc atccgcgagc acaaagcaac ggtgactctc gctgctgtga tgggtgcctt
     1801 catcatctgc tggttccctt actttaccgc ctttgtgtac cggggcctgc gaggcgacga
     1861 cgctattaac gaggtcctgg aggccatcgt cctgtggctg ggctacgcca acagtgctct
     1921 taacccaatc ctctatgcgg ccctcaaccg ggattttagg acagggtacc agcagctttt
     1981 ttgctgcagg cttgcaaatc ggaattccca taagacatcc ctccggagta acgccagtca
     2041 gctcagccga acacagagca gggagccgcg gcagcaggag gaaaaacctc ttaagctcca
     2101 ggtttggtca ggaacagagg tgactgcccc tcaaggtgct accgacagaa tcgataccgg
     2161 tggacgcacc ccacccagcc tgggtcccca agatgagtcc tgcaccaccg ccagctcctc
     2221 cctggccaag gacacttcat cgaccggtga gaacctgtac ttccagctaa gattagataa
     2281 aagtaaagtg attaacagcg cattagagct gcttaatgag gtcggaatcg aaggtttaac
     2341 aacccgtaaa ctcgcccaga agctaggtgt agagcagcct acattgtatt ggcatgtaaa
     2401 aaataagcgg gctttgctcg acgccttagc cattgagatg ttagataggc accatactca
     2461 cttttgccct ttagaagggg aaagctggca agatttttta cgtaataacg ctaaaagttt
     2521 tagatgtgct ttactaagtc atcgcgatgg agcaaaagta catttaggta cacggcctac
     2581 agaaaaacag tatgaaactc tcgaaaatca attagccttt ttatgccaac aaggtttttc
     2641 actagagaat gcattatatg cactcagcgc tgtggggcat tttactttag gttgcgtatt
     2701 ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa acacctacta ctgatagtat
     2761 gccgccatta ttacgacaag ctatcgaatt atttgatcac caaggtgcag agccagcctt
     2821 cttattcggc cttgaattga tcatatgcgg attagaaaaa caacttaaat gtgaaagtgg
     2881 gtccgcgtac agccgcgcgc gtacgaaaaa caattacggg tctaccatcg agggcctgct
     2941 cgatctcccg gacgacgacg cccccgaaga ggcggggctg gcggctccgc gcctgtcctt
     3001 tctccccgcg ggacacacgc gcagactgtc gacggccccc ccgaccgatg tcagcctggg
     3061 ggacgagctc cacttagacg gcgaggacgt ggcgatggcg catgccgacg cgctagacga
     3121 tttcgatctg gacatgttgg gggacgggga ttccccgggt ccgggattta ccccccacga
     3181 ctccgccccc tacggcgctc tggatatggc cgacttcgag tttgagcaga tgtttaccga
     3241 tgcccttgga attgacgagt acggtgggta gtctagaggg cccgtttaaa cccgctgatc
     3301 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3361 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3421 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3481 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     3541 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     3601 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3661 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3721 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3781 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3841 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3901 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3961 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     4021 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4081 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     4141 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4201 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4261 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     4321 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     4381 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     4441 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4501 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4561 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     4621 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4681 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4741 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4801 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4861 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4921 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4981 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     5041 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     5101 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     5161 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     5221 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     5281 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5341 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     5401 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5461 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5521 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5581 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5641 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5701 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5761 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5821 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5881 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5941 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6001 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6061 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6121 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6181 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6241 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6301 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6361 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6421 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6481 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6541 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6601 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6661 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6721 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6781 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6841 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6901 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6961 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7021 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7081 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7141 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7201 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7261 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7321 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7381 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7441 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7501 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7561 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7621 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7681 gcacatttcc ccgaaaagtg ccacctgacg tc
//
