LOCUS       Exported                7874 bp ds-DNA     circular SYN 12-MAY-2021
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    ADRB2-Tango
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7874)
  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 7874)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7874
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     865..884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        865..883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2434..3435
                     /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(2501..2520)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3467..3484)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3473..3697
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3743..4171
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3830..3849)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4040..4061
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4180..4200)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4185..4514
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4365..4500
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4427..4446
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4581..5375
                     /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(4635..4654)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5245..5264
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5549..5670
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5586..5605)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5640..5659
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5719..5735)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5719..5735)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5732..5754)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5743..5759
                     /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(5767..5797)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5812..5833
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5950..5967)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6121..6709)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6201..6220)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6880..7740)
                     /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     7503..7522
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7741..7845)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      841 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta
      901 gcgtttaaac ttaagcttgg taccgagctc ggatccacta gtccagtgtg gtggaattct
      961 gcagatatcc agcacagtgg cggccgcgcc accatgaaga cgatcatcgc cctgagctac
     1021 atcttctgcc tggtattcgc cgactacaag gacgatgatg acgccagcat cgatatgggg
     1081 caacctggca atggatcagc ttttctcctc gcccctaatc ggagccacgc tcccgaccac
     1141 gatgtgactc agcagagaga cgaggtctgg gttgtaggca tgggtattgt gatgtcactg
     1201 atcgtcctgg caattgtgtt cggcaacgtc ctcgtcatta ctgctattgc aaagttcgag
     1261 cggcttcaga cggttaccaa ttactttatt acttccctcg cctgtgccga cttggtcatg
     1321 ggccttgcgg ttgtaccgtt tggcgcagct catatcctga tgaagatgtg gacttttggt
     1381 aatttttggt gcgaattctg gacttcaatt gacgtgctct gcgtcacagc gtcaatcgaa
     1441 actctgtgtg ttattgccgt tgataggtac ttcgcgatta cgagtccctt taaataccag
     1501 tctctgctga ctaagaacaa agctcgggtt atcatactca tggtgtggat cgtctcagga
     1561 ctcacaagct ttctgcctat acagatgcac tggtatcgcg ccacacacca agaagctatc
     1621 aattgttacg caaacgaaac ctgttgcgac ttcttcacta atcaagccta tgctatcgct
     1681 agttcaattg tctcttttta tgtgcctctg gttatcatgg tgttcgtcta ctctcgggtt
     1741 tttcaggagg caaaaagaca gctccagaaa atcgacaagt cagaaggccg atttcacgtt
     1801 caaaatctga gccaggtcga acaagacgga agaactggac atggtcttcg gagatcttcc
     1861 aaattttgct tgaaggaaca caaggccctg aagaccttgg ggataattat gggcacattc
     1921 actctctgct ggctcccctt tttcattgtc aatatcgttc acgttataca agacaacttg
     1981 atcagaaaag aggtgtatat cctcctgaac tggattggct acgtgaactc tgggttcaac
     2041 cctctgattt actgccgcag cccagacttc agaatcgcat tccaggaatt gctgtgtctg
     2101 aggcgctctt ccttgaaggc ttatggaaac ggatactcct ctaatggcaa cacgggcgaa
     2161 cagtcaggat accacgtgga gcaggaaaaa gagaataagc tgttgtgcga ggacctccct
     2221 ggcactgagg attttgttgg ccaccaagga acagtcccaa gcgacaatat tgacagccag
     2281 gggcgaaact gcagcactaa tgattcactg ctgatcgata ccggtggacg caccccaccc
     2341 agcctgggtc cccaagatga gtcctgcacc accgccagct cctccctggc caaggacact
     2401 tcatcgaccg gtgagaacct gtacttccag ctaagattag ataaaagtaa agtgattaac
     2461 agcgcattag agctgcttaa tgaggtcgga atcgaaggtt taacaacccg taaactcgcc
     2521 cagaagctag gtgtagagca gcctacattg tattggcatg taaaaaataa gcgggctttg
     2581 ctcgacgcct tagccattga gatgttagat aggcaccata ctcacttttg ccctttagaa
     2641 ggggaaagct ggcaagattt tttacgtaat aacgctaaaa gttttagatg tgctttacta
     2701 agtcatcgcg atggagcaaa agtacattta ggtacacggc ctacagaaaa acagtatgaa
     2761 actctcgaaa atcaattagc ctttttatgc caacaaggtt tttcactaga gaatgcatta
     2821 tatgcactca gcgctgtggg gcattttact ttaggttgcg tattggaaga tcaagagcat
     2881 caagtcgcta aagaagaaag ggaaacacct actactgata gtatgccgcc attattacga
     2941 caagctatcg aattatttga tcaccaaggt gcagagccag ccttcttatt cggccttgaa
     3001 ttgatcatat gcggattaga aaaacaactt aaatgtgaaa gtgggtccgc gtacagccgc
     3061 gcgcgtacga aaaacaatta cgggtctacc atcgagggcc tgctcgatct cccggacgac
     3121 gacgcccccg aagaggcggg gctggcggct ccgcgcctgt cctttctccc cgcgggacac
     3181 acgcgcagac tgtcgacggc ccccccgacc gatgtcagcc tgggggacga gctccactta
     3241 gacggcgagg acgtggcgat ggcgcatgcc gacgcgctag acgatttcga tctggacatg
     3301 ttgggggacg gggattcccc gggtccggga tttacccccc acgactccgc cccctacggc
     3361 gctctggata tggccgactt cgagtttgag cagatgttta ccgatgccct tggaattgac
     3421 gagtacggtg ggtagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
     3481 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3541 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3601 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3661 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     3721 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3781 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3841 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3901 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3961 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4021 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     4081 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     4141 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     4201 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4261 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4321 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4381 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4441 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4501 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     4561 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     4621 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     4681 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     4741 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     4801 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     4861 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     4921 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     4981 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     5041 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     5101 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     5161 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     5221 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     5281 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     5341 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     5401 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     5461 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     5521 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     5581 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     5641 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     5701 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5761 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5821 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5881 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     5941 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     6001 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6061 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6121 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6181 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6241 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6301 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6361 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6421 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6481 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6541 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6601 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6661 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6721 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6781 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6841 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6901 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6961 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     7021 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7081 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7141 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7201 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7261 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7321 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7381 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7441 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7501 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7561 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7621 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     7681 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     7741 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     7801 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     7861 agtgccacct gacg
//
