LOCUS       sequence-485744-        7715 bp    DNA     circular SYN 10-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 7715)
  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 7715)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 10, 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: GPR15-Tango
FEATURES             Location/Qualifiers
     source          1..7715
                     /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(2039..2653,2654..3040)
                     /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(2106..2125)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3072..3089)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3078..3302
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3348..3776
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3435..3454)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3645..3666
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3785..3805)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3790..4119
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3970..4105
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4032..4051
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4186..4980
                     /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(4240..4259)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4850..4869
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5154..5275
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5191..5210)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5245..5264
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5324..5340)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5324..5340)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5337..5359)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5348..5364
                     /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(5372..5378,5379..5396,5397..5402))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5417..5438
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5555..5572)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5726..6314)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5806..5825)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6485..7276,7277..7345))
                     /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     7108..7127
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7346..7450)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7525..7544)
                     /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 ggatcctgag gagacaagcg tctacctgga ttactattat gccacctccc ccaacagtga
      901 catcagagag actcactccc atgtaccata cacatctgtc tttctgcctg tgttttacac
      961 cgccgtattc cttacaggcg ttctggggaa cctggtgctg atgggcgcct tgcacttcaa
     1021 gcctggtagt agacgcctga ttgatatatt tataattaac cttgcagcca gcgactttat
     1081 attcttggtg actttgcctc tgtgggttga taaggaggcc agtttggggt tgtggcggac
     1141 ggggagtttc ctgtgtaagg gtagcagtta tatgattagt gtaaatatgc attgtagtgt
     1201 tctgttgctg acgtgtatgt ctgtcgatcg ctatctggca atcgtgtggc ctgtggtgag
     1261 ccggaagttc cgccggaccg actgcgctta tgtcgtgtgc gcctctatct ggttcatttc
     1321 ttgcctgctg ggccttccca ccctcttgtc ccgggagctg acgctgattg atgacaagcc
     1381 ttactgcgcc gagaaaaaag ctaccccgat taaactgata tggtcactgg tggcgcttat
     1441 cttcacgttt tttgtgccgc tgctgtccat tgtcacctgt tattgctgta tcgctcggaa
     1501 gctttgcgcc cattatcagc aaagcggcaa acataataag aagctcaaga agtccattaa
     1561 aattatcttt atcgtcgtcg ctgcatttct ggtttcttgg ctgccattta atacattcaa
     1621 gtttctcgct atcgtgtctg gtcttagaca ggagcattac ctgcctagcg ctattctcca
     1681 actcggcatg gaggtaagcg gtccactggc tttcgcaaac tcatgtgtga accccttcat
     1741 ctattacatc ttcgattctt acattcggcg cgccattgtt cactgtctgt gtccttgtct
     1801 taaaaattat gactttggca gctctacaga gacatctgat tctcacctta ccaaggcctt
     1861 gtctactttt atccacgccg aggactttgc tcgaaggagg aagcgatccg taagcctcat
     1921 cgataccggt ggacgcaccc cacccagcct gggtccccaa gatgagtcct gcaccaccgc
     1981 cagctcctcc ctggccaagg acacttcatc gaccggtgag aacctgtact tccagctaag
     2041 attagataaa agtaaagtga ttaacagcgc attagagctg cttaatgagg tcggaatcga
     2101 aggtttaaca acccgtaaac tcgcccagaa gctaggtgta gagcagccta cattgtattg
     2161 gcatgtaaaa aataagcggg ctttgctcga cgccttagcc attgagatgt tagataggca
     2221 ccatactcac ttttgccctt tagaagggga aagctggcaa gattttttac gtaataacgc
     2281 taaaagtttt agatgtgctt tactaagtca tcgcgatgga gcaaaagtac atttaggtac
     2341 acggcctaca gaaaaacagt atgaaactct cgaaaatcaa ttagcctttt tatgccaaca
     2401 aggtttttca ctagagaatg cattatatgc actcagcgct gtggggcatt ttactttagg
     2461 ttgcgtattg gaagatcaag agcatcaagt cgctaaagaa gaaagggaaa cacctactac
     2521 tgatagtatg ccgccattat tacgacaagc tatcgaatta tttgatcacc aaggtgcaga
     2581 gccagccttc ttattcggcc ttgaattgat catatgcgga ttagaaaaac aacttaaatg
     2641 tgaaagtggg tccgcgtaca gccgcgcgcg tacgaaaaac aattacgggt ctaccatcga
     2701 gggcctgctc gatctcccgg acgacgacgc ccccgaagag gcggggctgg cggctccgcg
     2761 cctgtccttt ctccccgcgg gacacacgcg cagactgtcg acggcccccc cgaccgatgt
     2821 cagcctgggg gacgagctcc acttagacgg cgaggacgtg gcgatggcgc atgccgacgc
     2881 gctagacgat ttcgatctgg acatgttggg ggacggggat tccccgggtc cgggatttac
     2941 cccccacgac tccgccccct acggcgctct ggatatggcc gacttcgagt ttgagcagat
     3001 gtttaccgat gcccttggaa ttgacgagta cggtgggtag tctagagggc ccgtttaaac
     3061 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3121 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3181 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3241 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3301 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     3361 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3421 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3481 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3541 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3601 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3661 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3721 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     3781 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3841 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3901 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3961 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4021 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     4081 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     4141 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4201 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4261 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4321 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4381 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4441 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4501 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4561 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4621 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4681 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     4741 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4801 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4861 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4921 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4981 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     5041 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5101 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     5161 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5221 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5281 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     5341 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5401 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5461 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5521 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5581 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5641 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5701 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5761 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5821 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5881 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5941 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6001 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6061 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6121 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6181 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6241 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6301 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6361 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6421 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6481 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6541 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6601 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6661 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6721 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6781 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6841 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6901 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6961 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7021 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7081 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7141 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7201 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7261 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7321 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7381 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7441 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     7501 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     7561 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     7621 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     7681 gctgcttcgc gatgtacggg ccagatatac gcgtt
//
