LOCUS       sequence-372748-        7724 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 7724)
  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 7724)
  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: HCA2-Tango
FEATURES             Location/Qualifiers
     source          1..7724
                     /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(2048..2662,2663..3049)
                     /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(2115..2134)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3081..3098)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3087..3311
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3357..3785
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3444..3463)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3654..3675
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3794..3814)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3799..4128
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3979..4114
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4041..4060
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4195..4989
                     /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(4249..4268)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4859..4878
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5163..5284
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5200..5219)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5254..5273
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5333..5349)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5333..5349)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5346..5368)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5357..5373
                     /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(5381..5387,5388..5405,5406..5411))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5426..5447
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5564..5581)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5735..6323)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5815..5834)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6494..7285,7286..7354))
                     /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     7117..7136
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7355..7459)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7534..7553)
                     /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 gaaccggcac cacctgcagg atcacttcct ggaaatagac aaaaagaact gctgcgtttt
      901 tagagacgac tttatcgtca aagtactgcc cccagtgctc ggcctggagt tcatcttcgg
      961 acttctgggg aatggcctcg ccctctggat tttttgcttc caccttaagt cctggaagag
     1021 tagccgaatc ttcctgttca atctcgccgt agcggatttt ctgctcatca tttgcctgcc
     1081 tttcctgatg gacaattatg tgcgccggtg ggattggaaa ttcggtgata ttccttgtag
     1141 attgatgctc ttcatgctgg ctatgaaccg gcaaggaagc ataatctttc ttaccgttgt
     1201 ggccgtggac agatacttcc gagtggtgca tccacatcat gccctcaata agatttccaa
     1261 tcgaacggcc gctataatta gctgtcttct gtggggaatc accatcggcc tcaccgtcca
     1321 cctcctcaag aagaagatgc cgatccagaa cggtggggcc aacctgtgta gttcctttag
     1381 tatatgccat actttccagt ggcatgaggc tatgttcctg ctggagttct tcctgccact
     1441 tggcatcatc ttgttctgct ccgctcggat aatatggagc ctgagacaga gacagatgga
     1501 tcggcatgcg aaaataaagc gggccattac gttcatcatg gtcgtggcta ttgtgtttgt
     1561 gatttgtttc ctcccctctg tggtggtgag aataagaatt ttttggcttc tccacacgag
     1621 cggcacccaa aattgcgagg tttacaggag cgtggatctt gcctttttca tcactctgag
     1681 cttcacatac atgaattcaa tgctcgatcc agtggtttac tacttcagca gtccttcatt
     1741 cccgaacttt ttctctacgc tcatcaatcg gtgcctgcag cgcaaaatga ctggcgagcc
     1801 cgacaacaac agaagcacct ccgtggaact caccggcgac cctaacaaga cccggggtgc
     1861 ccccgaggct ctgatggcga actctggtga gccctggtcc cctagctatc tgggcccaac
     1921 gagccccatc gataccggtg gacgcacccc acccagcctg ggtccccaag atgagtcctg
     1981 caccaccgcc agctcctccc tggccaagga cacttcatcg accggtgaga acctgtactt
     2041 ccagctaaga ttagataaaa gtaaagtgat taacagcgca ttagagctgc ttaatgaggt
     2101 cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag ctaggtgtag agcagcctac
     2161 attgtattgg catgtaaaaa ataagcgggc tttgctcgac gccttagcca ttgagatgtt
     2221 agataggcac catactcact tttgcccttt agaaggggaa agctggcaag attttttacg
     2281 taataacgct aaaagtttta gatgtgcttt actaagtcat cgcgatggag caaaagtaca
     2341 tttaggtaca cggcctacag aaaaacagta tgaaactctc gaaaatcaat tagccttttt
     2401 atgccaacaa ggtttttcac tagagaatgc attatatgca ctcagcgctg tggggcattt
     2461 tactttaggt tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag aaagggaaac
     2521 acctactact gatagtatgc cgccattatt acgacaagct atcgaattat ttgatcacca
     2581 aggtgcagag ccagccttct tattcggcct tgaattgatc atatgcggat tagaaaaaca
     2641 acttaaatgt gaaagtgggt ccgcgtacag ccgcgcgcgt acgaaaaaca attacgggtc
     2701 taccatcgag ggcctgctcg atctcccgga cgacgacgcc cccgaagagg cggggctggc
     2761 ggctccgcgc ctgtcctttc tccccgcggg acacacgcgc agactgtcga cggccccccc
     2821 gaccgatgtc agcctggggg acgagctcca cttagacggc gaggacgtgg cgatggcgca
     2881 tgccgacgcg ctagacgatt tcgatctgga catgttgggg gacggggatt ccccgggtcc
     2941 gggatttacc ccccacgact ccgcccccta cggcgctctg gatatggccg acttcgagtt
     3001 tgagcagatg tttaccgatg cccttggaat tgacgagtac ggtgggtagt ctagagggcc
     3061 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3121 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3181 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3241 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3301 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3361 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3421 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3481 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3541 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3601 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3661 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3721 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3781 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3841 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3901 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3961 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4021 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     4081 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     4141 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4201 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4261 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4321 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     4381 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4441 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4501 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4561 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     4621 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     4681 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     4741 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4801 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4861 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4921 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4981 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     5041 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5101 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5161 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5221 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5281 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5341 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5401 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5461 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5521 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5581 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5641 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5701 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5761 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5821 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5881 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5941 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6001 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6061 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6121 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6181 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6241 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6301 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6361 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6421 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6481 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6541 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6601 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6661 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6721 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6781 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6841 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6901 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6961 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7021 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7081 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7141 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7201 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7261 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7321 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7381 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7441 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     7501 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     7561 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     7621 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     7681 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgtt
//
