LOCUS       sequence-299761-        7748 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 7748)
  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 7748)
  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: AVPR2-Tango
FEATURES             Location/Qualifiers
     source          1..7748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        7..386
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        387..590
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     541..561
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     635..654
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        635..653
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(2078..2692,2693..3079)
                     /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(2145..2164)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3111..3128)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3117..3341
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3387..3815
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3474..3493)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3684..3705
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3824..3844)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3829..4158
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4009..4144
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4071..4090
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4225..5019
                     /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(4279..4298)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4889..4908
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5193..5314
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5230..5249)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5284..5303
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5363..5379)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5363..5379)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5376..5398)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5387..5403
                     /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(5411..5417,5418..5435,5436..5441))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5456..5477
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5594..5611)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5765..6353)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5845..5864)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6524..7315,7316..7384))
                     /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     7147..7166
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7385..7489)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7564..7583)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
       61 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      121 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      181 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      241 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      301 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      361 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      421 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      481 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      541 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
      601 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
      661 aagctggcta gcgtttaaac ttaagcttgg taccgagctc ggatccacta gtccagtgtg
      721 gtggaattct gcagatatcc agcacagtgg cggccgcgcc accatgaaga cgatcatcgc
      781 cctgagctac atcttctgcc tggtattcgc cgactacaag gacgatgatg acgccagcat
      841 cgatatgctc atggcctcta ccacaagcgc tgtgcccgga catccatctc ttccaagtct
      901 tccctccaac agctcacagg agaggcctct ggatacccgg gacccacttc ttgcaagagc
      961 ggagctcgcc ttgctctcca tcgttttcgt tgccgtcgcg ttgagcaatg gtctggtgct
     1021 ggccgccctg gcgcgacgag gccgccgagg ccactgggcg cctattcatg tctttatcgg
     1081 ccatctctgt cttgcagacc tggccgtggc gctgtttcag gtgctccctc aactggcttg
     1141 gaaagccacc gatagattcc gggggccaga cgcactgtgt cgggccgtga aatacctgca
     1201 gatggtggga atgtatgcca gttcatatat gatacttgct atgaccttgg acagacacag
     1261 ggctatctgt cgccccatgc tggcgtatag gcacggttcc ggagcccatt ggaacaggcc
     1321 agttctggtt gcgtgggctt tttccctgct tctgagcttg ccccagttgt ttattttcgc
     1381 acagagaaat gtggagggcg gatctggcgt gaccgattgc tgggcttgct tcgctgagcc
     1441 atggggcaga aggacctacg tgacgtggat tgctctcatg gtgttcgttg ctcccaccct
     1501 ggggatcgcc gcatgccagg tcctgatatt cagagaaata cacgcctcct tggttcctgg
     1561 gccaagcgag cgccctggtg gaagaagacg gggtcgaagg actggctctc caggcgaagg
     1621 cgcccacgta tcagctgcgg tagctaagac cgtacgcatg acactggtca tcgtcgtcgt
     1681 ttacgttctt tgctgggccc cgtttttttt ggtgcagctc tgggcggcct gggaccccga
     1741 agcacccctg gaaggtgctc cattcgttct gctcatgctt ctcgcttcac tgaatagctg
     1801 taccaatcct tggatttacg cctctttctc ctccagtgtt agctccgagc tgcggagtct
     1861 tctctgttgc gcccggggca ggaccccccc aagcctgggt ccacaggacg aatcctgcac
     1921 taccgcctcc agttccctgg ccaaagacac ctcttctatc gataccggtg gacgcacccc
     1981 acccagcctg ggtccccaag atgagtcctg caccaccgcc agctcctccc tggccaagga
     2041 cacttcatcg accggtgaga acctgtactt ccagctaaga ttagataaaa gtaaagtgat
     2101 taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact
     2161 cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa ataagcgggc
     2221 tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact tttgcccttt
     2281 agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta gatgtgcttt
     2341 actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag aaaaacagta
     2401 tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac tagagaatgc
     2461 attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg aagatcaaga
     2521 gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc cgccattatt
     2581 acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct tattcggcct
     2641 tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt ccgcgtacag
     2701 ccgcgcgcgt acgaaaaaca attacgggtc taccatcgag ggcctgctcg atctcccgga
     2761 cgacgacgcc cccgaagagg cggggctggc ggctccgcgc ctgtcctttc tccccgcggg
     2821 acacacgcgc agactgtcga cggccccccc gaccgatgtc agcctggggg acgagctcca
     2881 cttagacggc gaggacgtgg cgatggcgca tgccgacgcg ctagacgatt tcgatctgga
     2941 catgttgggg gacggggatt ccccgggtcc gggatttacc ccccacgact ccgcccccta
     3001 cggcgctctg gatatggccg acttcgagtt tgagcagatg tttaccgatg cccttggaat
     3061 tgacgagtac ggtgggtagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     3121 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3181 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3241 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3301 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3361 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3421 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3481 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3541 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3601 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3661 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3721 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3781 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3841 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3901 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3961 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4021 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4081 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4141 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     4201 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     4261 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4321 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4381 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4441 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4501 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4561 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4621 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4681 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4741 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4801 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4861 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4921 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4981 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     5041 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     5101 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5161 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     5221 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5281 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     5341 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5401 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5461 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5521 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5581 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5641 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5701 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5761 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5821 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5881 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5941 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6001 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6061 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6121 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6181 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6241 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6301 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6361 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6421 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6481 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6541 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6601 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6661 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6721 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6781 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6841 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6901 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6961 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7021 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7081 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7141 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7201 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7261 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7321 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7381 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7441 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7501 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     7561 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     7621 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     7681 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     7741 cagatata
//
