LOCUS       sequence-321941-        8219 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 8219)
  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 8219)
  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: GPR114-Tango
FEATURES             Location/Qualifiers
     source          1..8219
                     /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(2543..3157,3158..3544)
                     /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(2610..2629)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3576..3593)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3582..3806
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3852..4280
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3939..3958)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4149..4170
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4289..4309)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4294..4623
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4474..4609
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4536..4555
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4690..5484
                     /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(4744..4763)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5354..5373
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5658..5779
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5695..5714)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5749..5768
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5828..5844)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5828..5844)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5841..5863)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5852..5868
                     /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(5876..5882,5883..5900,5901..5906))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5921..5942
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6059..6076)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6230..6818)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6310..6329)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6989..7780,7781..7849))
                     /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     7612..7631
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7850..7954)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8029..8048)
                     /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 ggatcattgc ggagcgctgt tcttgtgtct gtgtctcctc actctgcaga atgccacgac
      901 cgagacctgg gaggaactcc tcagctacat ggagaatatg caggttagca gaggcaggtc
      961 ctctgtgttc agctctagac agctgcacca gcttgagcag atgctcctta ataccagctt
     1021 ccctggctac aatctcaccc tgcagactcc aacgattcag tcactggcat ttaagctgag
     1081 ctgcgacttt tccggcctga gcctgacttc cgcgacactc aaacgagtcc cgcaggctgg
     1141 aggccagcac gcacgcggtc agcatgccat gcagttcccc gcggaactta ctcgcgacgc
     1201 gtgtaagaca cgacccaggg aactgaggct gatctgcatt tatttttcca atacacattt
     1261 ttttaaagac gaaaataatt ctagcttgct gaacaattac gtgctgggag cccagctcag
     1321 tcatggccac gtgaataacc ttcgagatcc cgttaacata tccttttggc ataaccagag
     1381 tctggaaggg tacacactga cctgcgtgtt ctggaaggag ggggctagga aacaaccatg
     1441 gggcggatgg tcccctgagg gttgccgcac agagcagcca agtcactctc aggtgctgtg
     1501 ccgctgcaac catctcacat acttcgccgt gctcatgcag ctttcacccg ctctcgtccc
     1561 tgccgaactt ctcgcccccc tgacgtatat ttcactcgtg ggctgtagca tcagcatagt
     1621 cgcctctctg atcaccgtgc tgctgcactt ccattttaga aagcagagcg actctctgac
     1681 ccgcatccac atgaatctcc acgcaagcgt cctgctgctc aatattgctt tcttgctgtc
     1741 acccgctttc gctatgtccc ctgtgccagg gagtgcttgc actgccctcg cggcagcgct
     1801 ccactatgcc ctcctgtctt gtctgacttg gatggcaatc gagggattta acctctacct
     1861 cctgctgggg agagtataca atatctacat ccggagatac gtgttcaagc tgggcgttct
     1921 tggctggggc gcccctgcgc tgctcgtgct gttgtcactg tccgtgaaat catctgtata
     1981 tggcccatgt actattcctg ttttcgacag ctgggagaac ggaactgggt tccagaatat
     2041 gtccatctgc tgggttcgca gcccagtggt tcactcagtg ctggttatgg gatacggagg
     2101 actgacctct ctcttcaact tggtggtgct ggcatgggcc ctgtggaccc ttcggcgact
     2161 gcgggagaga gccgacgccc ccagcgtaag agcttgccac gacaccgtga cagtcctcgg
     2221 actgacggtt ctgctcggta caacgtgggc actcgctttt ttcagcttcg gcgtgtttct
     2281 gcttccacag ctgtttcttt tcactatcct gaattctctc tacggctttt tcctctttct
     2341 gtggttttgc tctcagcgct gccgctccga ggcagaggcg aaggcccaaa tcgaggcttt
     2401 ctccagtagc cagactactc aaatcgatac cggtggacgc accccaccca gcctgggtcc
     2461 ccaagatgag tcctgcacca ccgccagctc ctccctggcc aaggacactt catcgaccgg
     2521 tgagaacctg tacttccagc taagattaga taaaagtaaa gtgattaaca gcgcattaga
     2581 gctgcttaat gaggtcggaa tcgaaggttt aacaacccgt aaactcgccc agaagctagg
     2641 tgtagagcag cctacattgt attggcatgt aaaaaataag cgggctttgc tcgacgcctt
     2701 agccattgag atgttagata ggcaccatac tcacttttgc cctttagaag gggaaagctg
     2761 gcaagatttt ttacgtaata acgctaaaag ttttagatgt gctttactaa gtcatcgcga
     2821 tggagcaaaa gtacatttag gtacacggcc tacagaaaaa cagtatgaaa ctctcgaaaa
     2881 tcaattagcc tttttatgcc aacaaggttt ttcactagag aatgcattat atgcactcag
     2941 cgctgtgggg cattttactt taggttgcgt attggaagat caagagcatc aagtcgctaa
     3001 agaagaaagg gaaacaccta ctactgatag tatgccgcca ttattacgac aagctatcga
     3061 attatttgat caccaaggtg cagagccagc cttcttattc ggccttgaat tgatcatatg
     3121 cggattagaa aaacaactta aatgtgaaag tgggtccgcg tacagccgcg cgcgtacgaa
     3181 aaacaattac gggtctacca tcgagggcct gctcgatctc ccggacgacg acgcccccga
     3241 agaggcgggg ctggcggctc cgcgcctgtc ctttctcccc gcgggacaca cgcgcagact
     3301 gtcgacggcc cccccgaccg atgtcagcct gggggacgag ctccacttag acggcgagga
     3361 cgtggcgatg gcgcatgccg acgcgctaga cgatttcgat ctggacatgt tgggggacgg
     3421 ggattccccg ggtccgggat ttacccccca cgactccgcc ccctacggcg ctctggatat
     3481 ggccgacttc gagtttgagc agatgtttac cgatgccctt ggaattgacg agtacggtgg
     3541 gtagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     3601 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3661 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3721 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3781 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     3841 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3901 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3961 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4021 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4081 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4141 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4201 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4261 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     4321 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     4381 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4441 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4501 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4561 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4621 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     4681 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4741 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4801 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4861 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4921 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     4981 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     5041 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     5101 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     5161 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     5221 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     5281 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     5341 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     5401 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     5461 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     5521 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5581 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5641 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5701 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5761 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     5821 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5881 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5941 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6001 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     6061 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6121 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6181 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6241 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6301 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6361 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6421 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6481 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6541 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6601 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6661 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6721 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6781 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6841 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6901 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6961 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     7021 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     7081 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     7141 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7201 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7261 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7321 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7381 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7441 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7501 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7561 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7621 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7681 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7741 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7801 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7861 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7921 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7981 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
     8041 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     8101 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     8161 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgtt
//
