LOCUS        CHRM2-Tango       8033 bp    DNA     circular SYN 14-APR-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 8033)
  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 8033)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
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: CHRM2-Tango
FEATURES             Location/Qualifiers
     source          1..8033
                     /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(2357..2971,2972..3358)
                     /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(2424..2443)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3390..3407)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3396..3620
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3666..4094
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3753..3772)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3963..3984
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4103..4123)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4108..4437
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4288..4423
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4350..4369
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4504..5298
                     /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(4558..4577)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5168..5187
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5472..5593
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5509..5528)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5563..5582
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5642..5658)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5642..5658)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5655..5677)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5666..5682
                     /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(5690..5696,5697..5714,5715..5720))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5735..5756
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5873..5890)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6044..6632)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6124..6143)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6803..7594,7595..7663))
                     /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     7426..7445
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7664..7768)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7843..7862)
                     /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 gaacaatagt actaatagct caaacaactc attggccctc acaagccctt acaagacctt
      901 tgaggttgtc ttcattgtgc tggtggctgg atcactgtcc ctggtaacca tcatcggcaa
      961 tatccttgtg atggtgtcta taaaggtcaa taggcacttg cagacagtca acaactattt
     1021 cctgttctca ttggcatgcg cagatttgat aataggcgta ttctccatga acctttatac
     1081 actgtatact gtcattggat attggccatt gggccccgtg gtctgcgacc tttggcttgc
     1141 actcgactac gttgtttcca atgcatctgt aatgaacctg ctgattatct cattcgatcg
     1201 gtacttctgc gttaccaagc ccctgactta cccagtgaag aggactacaa agatggccgg
     1261 aatgatgatt gcagcagctt gggtgctttc ttttatactg tgggctcccg ctattctgtt
     1321 ctggcagttt attgtgggtg taaggactgt ggaagacggc gagtgctata tccagttctt
     1381 ctctaacgca gctgtaacct tcgggactgc catcgctgcc ttctacctgc ccgtcatcat
     1441 tatgacagtg ctgtattggc atatctcaag agctagcaag tcccggataa aaaaggacaa
     1501 gaaagagcca gttgcaaatc aagaccccgt ctcaccttcc cttgtgcagg ggagaatcgt
     1561 caaaccaaat aataacaata tgccgagctc cgacgacggc ctggaacata acaaaattca
     1621 gaatgggaaa gcaccaaggg accccgtgac cgagaattgc gtgcaggggg aggagaaaga
     1681 gagttctaac gactctacct ctgtcagcgc cgtggcttcc aatatgcggg atgatgaaat
     1741 tactcaggat gaaaatactg tttccacatc cttgggccac agcaaggacg aaaactccaa
     1801 gcaaacatgc attcgcattg ggaccaagac acctaaatcc gacagttgca ctccgacaaa
     1861 cacaactgtc gaagtggtgg gttcctcagg ccagaacgga gacgagaagc agaacattgt
     1921 tgcccggaag atcgtgaaga tgaccaaaca gcccgctaag aaaaagcctc cccccagtcg
     1981 cgaaaaaaag gtgactagaa ctatcctcgc tatcctcctg gcattcatta tcacatgggc
     2041 cccatacaat gttatggtac ttatcaacac tttctgcgct ccctgtatcc ccaacactgt
     2101 ttggactatc ggctactggc tgtgttatat taacagtaca atcaaccctg catgctatgc
     2161 cctgtgtaat gccacattca aaaaaacatt taagcacctc ctgatgtgtc actacaaaaa
     2221 cataggcgcg acacgcatcg ataccggtgg acgcacccca cccagcctgg gtccccaaga
     2281 tgagtcctgc accaccgcca gctcctccct ggccaaggac acttcatcga ccggtgagaa
     2341 cctgtacttc cagctaagat tagataaaag taaagtgatt aacagcgcat tagagctgct
     2401 taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga
     2461 gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat
     2521 tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga
     2581 ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc
     2641 aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt
     2701 agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt
     2761 ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga
     2821 aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt
     2881 tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt
     2941 agaaaaacaa cttaaatgtg aaagtgggtc cgcgtacagc cgcgcgcgta cgaaaaacaa
     3001 ttacgggtct accatcgagg gcctgctcga tctcccggac gacgacgccc ccgaagaggc
     3061 ggggctggcg gctccgcgcc tgtcctttct ccccgcggga cacacgcgca gactgtcgac
     3121 ggcccccccg accgatgtca gcctggggga cgagctccac ttagacggcg aggacgtggc
     3181 gatggcgcat gccgacgcgc tagacgattt cgatctggac atgttggggg acggggattc
     3241 cccgggtccg ggatttaccc cccacgactc cgccccctac ggcgctctgg atatggccga
     3301 cttcgagttt gagcagatgt ttaccgatgc ccttggaatt gacgagtacg gtgggtagtc
     3361 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     3421 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3481 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3541 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3601 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     3661 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3721 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3781 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3841 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3901 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3961 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     4021 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4081 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     4141 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     4201 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4261 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4321 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     4381 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     4441 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     4501 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     4561 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     4621 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     4681 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     4741 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     4801 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     4861 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     4921 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4981 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     5041 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     5101 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     5161 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     5221 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     5281 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     5341 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     5401 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     5461 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     5521 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     5581 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     5641 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5701 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     5761 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5821 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5881 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5941 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6001 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6061 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6121 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6181 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6241 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6301 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6361 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6421 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6481 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6541 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6601 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6661 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6721 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6781 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6841 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6901 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6961 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7021 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7081 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7141 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7201 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7261 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7321 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7381 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7441 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7501 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7561 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7621 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7681 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7741 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     7801 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     7861 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     7921 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     7981 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//