LOCUS       Exported                7916 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    EDNRA-Tango
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7916)
  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 7916)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7916
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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             2240..3241
                     /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(2307..2326)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3273..3290)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3279..3503
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3549..3977
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3636..3655)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3846..3867
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3986..4006)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3991..4320
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4171..4306
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4233..4252
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4387..5181
                     /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(4441..4460)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5051..5070
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5355..5476
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5392..5411)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5446..5465
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5525..5541)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5525..5541)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5538..5560)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5549..5565
                     /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(5573..5603)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5618..5639
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5756..5773)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5927..6515)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6007..6026)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6686..7546)
                     /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     7309..7328
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7547..7651)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7726..7745)
                     /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 ggagacactg tgtctcaggg cctccttctg gctcgcactg gtagggtgtg tcatatctga
      901 taaccctgaa cggtacagta ccaatctgag taatcatgtc gatgacttta caacgtttcg
      961 gggcactgag ctgagctttc tcgtgactac gcaccagccc accaatctcg tcctgcctag
     1021 caacgggagt atgcacaact actgccccca gcagacgaaa ataacaagcg ccttcaagta
     1081 tatcaacacc gtgatatcct gcactatctt catagttggc atggtgggga acgcgaccct
     1141 gctccgcatt atttatcaga acaagtgtat gagaaacgga ccgaatgcac ttatcgcaag
     1201 tctggcactc ggagacctga tctacgtcgt cattgatctg ccaataaacg tgtttaagct
     1261 cctggccggc cgctggccct tcgatcacaa cgattttggg gtgtttcttt gcaagctctt
     1321 tccttttttg cagaaatcat ccgtgggcat caccgttctc aatctgtgtg cgctgtccgt
     1381 tgaccgatat cgcgccgtcg caagctggtc ccgggtccag ggaatcggaa tcccactggt
     1441 gaccgctatc gagatcgtgt caatctggat actgtccttt atcctcgcga ttccggaggc
     1501 aatcgggttt gtgatggtcc ctttcgagta tcgaggcgag cagcataaaa cctgcatgtt
     1561 gaatgctaca tcaaagttca tggagtttta tcaggatgtg aaagattggt ggcttttcgg
     1621 attctatttt tgcatgccct tggtttgtac cgcaattttt tatactctga tgacatgcga
     1681 gatgctcaat aggagaaacg gatctctgag gattgcactg tccgagcacc tgaaacagcg
     1741 gcgggaggtt gcgaagactg tcttctgtct ggtcgtgatc ttcgctctgt gctggttccc
     1801 cctgcatctg agccgcattc tcaagaaaac cgtctacaac gagatggaca aaaatagatg
     1861 cgaattgctc tcctttttgc ttttgatgga ctatattggg attaaccttg ccaccatgaa
     1921 ttcctgtatc aacccaattg ctctctactt tgtgtcaaag aaattcaaga actgtttcca
     1981 aagctgtctc tgttgctgtt gctaccagtc taagagtctg atgacctcag tgcccatgaa
     2041 tgggacaagt atccagtgga agaaccatga ccagaataac cacaacacag accgctcatc
     2101 ccataaggat agcatgaaca tcgataccgg tggacgcacc ccacccagcc tgggtcccca
     2161 agatgagtcc tgcaccaccg ccagctcctc cctggccaag gacacttcat cgaccggtga
     2221 gaacctgtac ttccagctaa gattagataa aagtaaagtg attaacagcg cattagagct
     2281 gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt
     2341 agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc
     2401 cattgagatg ttagataggc accatactca cttttgccct ttagaagggg aaagctggca
     2461 agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg
     2521 agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca
     2581 attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc
     2641 tgtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga
     2701 agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt
     2761 atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg
     2821 attagaaaaa caacttaaat gtgaaagtgg gtccgcgtac agccgcgcgc gtacgaaaaa
     2881 caattacggg tctaccatcg agggcctgct cgatctcccg gacgacgacg cccccgaaga
     2941 ggcggggctg gcggctccgc gcctgtcctt tctccccgcg ggacacacgc gcagactgtc
     3001 gacggccccc ccgaccgatg tcagcctggg ggacgagctc cacttagacg gcgaggacgt
     3061 ggcgatggcg catgccgacg cgctagacga tttcgatctg gacatgttgg gggacgggga
     3121 ttccccgggt ccgggattta ccccccacga ctccgccccc tacggcgctc tggatatggc
     3181 cgacttcgag tttgagcaga tgtttaccga tgcccttgga attgacgagt acggtgggta
     3241 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     3301 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3361 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3421 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3481 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3541 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3601 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3661 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3721 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3781 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3841 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3901 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3961 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     4021 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     4081 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4141 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4201 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     4261 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4321 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     4381 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4441 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4501 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4561 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4621 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4681 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4741 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4801 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4861 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4921 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4981 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     5041 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     5101 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     5161 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     5221 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5281 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5341 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5401 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5461 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     5521 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5581 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5641 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5701 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5761 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5821 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5881 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5941 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     6001 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6061 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6121 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6181 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6241 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6301 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6361 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6421 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6481 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6541 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6601 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6661 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6721 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6781 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6841 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6901 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6961 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     7021 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     7081 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     7141 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7201 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7261 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7321 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7381 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7441 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7501 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7561 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7621 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     7681 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     7741 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     7801 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     7861 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgtt
//
