LOCUS       Exported                7742 bp ds-DNA     circular SYN 15-OCT-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    SSTR2-Tango
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7742)
  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 7742)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 15, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7742
                     /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             2066..3067
                     /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(2133..2152)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3099..3116)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3105..3329
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3375..3803
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3462..3481)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3672..3693
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3812..3832)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3817..4146
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3997..4132
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4059..4078
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4213..5007
                     /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(4267..4286)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4877..4896
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5181..5302
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5218..5237)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5272..5291
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5351..5367)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5351..5367)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5364..5386)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5375..5391
                     /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(5399..5429)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5444..5465
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5582..5599)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5753..6341)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5833..5852)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6512..7372)
                     /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     7135..7154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7373..7477)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7552..7571)
                     /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 ggacatggcc gatgagcctc tgaacggttc ccacacctgg ctgagcatac ctttcgattt
      901 gaatggttca gtcgtgtcaa cgaacacgag caatcagacc gaaccatatt atgacctgac
      961 tagcaatgcc gttttgactt tcatttactt tgtggtgtgc atcatcggat tgtgcggaaa
     1021 taccctcgtc atttacgtca tcctgcggta tgcaaagatg aaaactatta ccaacattta
     1081 catcctcaat ctggccatcg cggatgaact gtttatgctg ggactgcctt tcctggcgat
     1141 gcaggtggca ctcgttcact ggcctttcgg aaaagctata tgtagggtcg taatgactgt
     1201 agacggaatt aaccagttca cttcaatttt ctgtttgact gttatgagta ttgaccgcta
     1261 tcttgccgtc gtccacccga taaagtcagc caagtggcgg agaccaagaa ccgctaaaat
     1321 gatcacaatg gctgtgtggg gggttagcct gctggttatc ctgccaatta tgatctatgc
     1381 aggccttagg tcaaatcaat ggggccgatc tagttgcaca atcaattggc ccggcgagag
     1441 tggagcatgg tacacggggt tcattatcta tacattcatt ctcggcttcc tggtaccact
     1501 cacaattata tgtctgtgtt acctgtttat aattattaag gtaaaaagca gcgggatacg
     1561 ggtgggaagt tcaaaacgaa agaagtctga aaaaaaagtg accagaatgg tgtctatcgt
     1621 ggtggctgta tttatttttt gttggttgcc gttctacatt ttcaatgtca gttcagtttc
     1681 tatggccata agcccaaccc ctgcacttaa aggtatgttt gatttcgtgg tcgttctgac
     1741 ctacgccaac agttgcgcca atcccattct gtacgccttt cttagcgaca acttcaagaa
     1801 aagctttcaa aacgttttgt gcctggttaa ggtgagtgga actgatgacg gcgagagatc
     1861 cgacagtaag caggacaaaa gtagacttaa tgagaccacc gagacccaac ggaccctcct
     1921 caatggggac ctgcagacct ccatcatcga taccggtgga cgcaccccac ccagcctggg
     1981 tccccaagat gagtcctgca ccaccgccag ctcctccctg gccaaggaca cttcatcgac
     2041 cggtgagaac ctgtacttcc agctaagatt agataaaagt aaagtgatta acagcgcatt
     2101 agagctgctt aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg cccagaagct
     2161 aggtgtagag cagcctacat tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc
     2221 cttagccatt gagatgttag ataggcacca tactcacttt tgccctttag aaggggaaag
     2281 ctggcaagat tttttacgta ataacgctaa aagttttaga tgtgctttac taagtcatcg
     2341 cgatggagca aaagtacatt taggtacacg gcctacagaa aaacagtatg aaactctcga
     2401 aaatcaatta gcctttttat gccaacaagg tttttcacta gagaatgcat tatatgcact
     2461 cagcgctgtg gggcatttta ctttaggttg cgtattggaa gatcaagagc atcaagtcgc
     2521 taaagaagaa agggaaacac ctactactga tagtatgccg ccattattac gacaagctat
     2581 cgaattattt gatcaccaag gtgcagagcc agccttctta ttcggccttg aattgatcat
     2641 atgcggatta gaaaaacaac ttaaatgtga aagtgggtcc gcgtacagcc gcgcgcgtac
     2701 gaaaaacaat tacgggtcta ccatcgaggg cctgctcgat ctcccggacg acgacgcccc
     2761 cgaagaggcg gggctggcgg ctccgcgcct gtcctttctc cccgcgggac acacgcgcag
     2821 actgtcgacg gcccccccga ccgatgtcag cctgggggac gagctccact tagacggcga
     2881 ggacgtggcg atggcgcatg ccgacgcgct agacgatttc gatctggaca tgttggggga
     2941 cggggattcc ccgggtccgg gatttacccc ccacgactcc gccccctacg gcgctctgga
     3001 tatggccgac ttcgagtttg agcagatgtt taccgatgcc cttggaattg acgagtacgg
     3061 tgggtagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     3121 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3181 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3241 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3301 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3361 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3421 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3481 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3541 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3601 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3661 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3721 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3781 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3841 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3901 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3961 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4021 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4081 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4141 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     4201 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4261 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4321 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4381 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4441 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4501 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4561 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4621 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4681 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4741 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4801 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4861 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4921 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4981 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     5041 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5101 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5161 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     5221 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5281 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5341 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5401 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5461 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5521 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5581 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5641 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5701 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5761 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5821 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5881 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5941 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6001 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6061 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6121 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6181 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6241 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6301 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6361 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6421 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6481 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6541 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6601 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6661 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6721 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6781 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6841 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6901 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6961 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7021 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7081 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7141 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7201 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7261 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7321 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7381 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7441 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7501 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     7561 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     7621 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     7681 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
     7741 tt
//
