LOCUS       Exported                8099 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    PTGER4-Tango
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8099)
  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 8099)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8099
                     /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             2423..3424
                     /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(2490..2509)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3456..3473)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3462..3686
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3732..4160
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3819..3838)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4029..4050
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4169..4189)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4174..4503
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4354..4489
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4416..4435
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4570..5364
                     /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(4624..4643)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5234..5253
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5538..5659
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5575..5594)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5629..5648
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5708..5724)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5708..5724)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5721..5743)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5732..5748
                     /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(5756..5786)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5801..5822
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5939..5956)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6110..6698)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6190..6209)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6869..7729)
                     /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     7492..7511
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7730..7834)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7909..7928)
                     /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 gtccacccca ggggtgaaca gtagtgctag tttgagccca gataggctta acagtcctgt
      901 aaccatcccg gctgttatgt ttatattcgg tgtagtgggc aatctggttg ccattgttgt
      961 gctgtgcaaa tctcgcaagg agcagaagga aactaccttt tacactctcg tctgtgggtt
     1021 ggccgtcact gacctgttgg ggacattgct ggtgtcacca gtgactatag caacgtacat
     1081 gaaaggtcag tggccaggag gtcagcccct gtgtgagtac agcaccttca ttttgctgtt
     1141 tttttctctc tccgggctgt caatcatctg tgctatgtcc gtggagcgct atctcgccat
     1201 taatcatgcc tacttctata gccattacgt agataagcgc ctcgccggct tgactttgtt
     1261 cgccgtgtat gcctcaaacg tgctgttttg tgccctgcct aacatgggcc tcggaagttc
     1321 ccgcctgcaa tacccagata catggtgctt tattgattgg acaactaacg ttactgctca
     1381 cgctgcatac tcatatatgt atgcagggtt tagttccttc ttgatcctgg ccaccgtgtt
     1441 gtgtaatgtg ctggtttgtg gtgccctctt gcgcatgcat cgacagttca tgaggcgcac
     1501 ctctctcggt acggaacagc atcacgcagc agccgctgcc tctgtggcct ccaggggaca
     1561 ccctgcagcc tcaccagcgc ttccgcggct gtccgatttc aggcggagac gatcattccg
     1621 cagaattgcc ggggctgaga tccagatggt cattcttctg attgccacca gtctggtagt
     1681 gctcatctgt tctattccac ttgtggttcg cgtcttcgtt aatcagctct accagccaag
     1741 tcttgagcgg gaggttagta agaacccgga tctccaggcc atcagaattg cctcagtgaa
     1801 tcctatcctg gacccctgga tctatattct gctgaggaaa acggtgctca gtaaggcaat
     1861 tgaaaagatt aaatgcctct tctgcagaat cggtgggagc aggcgggaaa gaagcgggca
     1921 gcattgtagc gattcccagc gaacgtcatc tgcaatgtct ggtcatagca ggtcctttat
     1981 cagcagggaa ctgaaggaaa ttagttctac atctcagaca ctgctccccg atctgagtct
     2041 gccggatctg agcgaaaatg ggctgggtgg tagaaatctg ttgccgggcg tgcccggtat
     2101 gggattggct caggaagata ccaccagcct gcgcacactt cgaatctctg agactagtga
     2161 cagcagtcag gggcaggact cagagtctgt gctgctggtg gacgaagccg gagggtccgg
     2221 tagggctgga cccgcaccga agggcagcag tcttcaggtg accttcccca gcgagacact
     2281 caatctcagc gagaagtgca ttatcgatac cggtggacgc accccaccca gcctgggtcc
     2341 ccaagatgag tcctgcacca ccgccagctc ctccctggcc aaggacactt catcgaccgg
     2401 tgagaacctg tacttccagc taagattaga taaaagtaaa gtgattaaca gcgcattaga
     2461 gctgcttaat gaggtcggaa tcgaaggttt aacaacccgt aaactcgccc agaagctagg
     2521 tgtagagcag cctacattgt attggcatgt aaaaaataag cgggctttgc tcgacgcctt
     2581 agccattgag atgttagata ggcaccatac tcacttttgc cctttagaag gggaaagctg
     2641 gcaagatttt ttacgtaata acgctaaaag ttttagatgt gctttactaa gtcatcgcga
     2701 tggagcaaaa gtacatttag gtacacggcc tacagaaaaa cagtatgaaa ctctcgaaaa
     2761 tcaattagcc tttttatgcc aacaaggttt ttcactagag aatgcattat atgcactcag
     2821 cgctgtgggg cattttactt taggttgcgt attggaagat caagagcatc aagtcgctaa
     2881 agaagaaagg gaaacaccta ctactgatag tatgccgcca ttattacgac aagctatcga
     2941 attatttgat caccaaggtg cagagccagc cttcttattc ggccttgaat tgatcatatg
     3001 cggattagaa aaacaactta aatgtgaaag tgggtccgcg tacagccgcg cgcgtacgaa
     3061 aaacaattac gggtctacca tcgagggcct gctcgatctc ccggacgacg acgcccccga
     3121 agaggcgggg ctggcggctc cgcgcctgtc ctttctcccc gcgggacaca cgcgcagact
     3181 gtcgacggcc cccccgaccg atgtcagcct gggggacgag ctccacttag acggcgagga
     3241 cgtggcgatg gcgcatgccg acgcgctaga cgatttcgat ctggacatgt tgggggacgg
     3301 ggattccccg ggtccgggat ttacccccca cgactccgcc ccctacggcg ctctggatat
     3361 ggccgacttc gagtttgagc agatgtttac cgatgccctt ggaattgacg agtacggtgg
     3421 gtagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     3481 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3541 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3601 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3661 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     3721 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3781 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3841 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3901 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3961 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4021 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4081 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4141 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     4201 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     4261 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4321 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4381 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4441 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4501 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     4561 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4621 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4681 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4741 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4801 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     4861 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     4921 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     4981 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     5041 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     5101 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     5161 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     5221 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     5281 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     5341 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     5401 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5461 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5521 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5581 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5641 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     5701 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5761 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5821 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5881 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5941 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6001 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6061 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6121 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6181 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6241 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6301 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6361 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6421 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6481 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6541 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6601 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6661 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6721 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6781 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6841 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6901 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6961 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     7021 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7081 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7141 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7201 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7261 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7321 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7381 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7441 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7501 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7561 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7621 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7681 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7741 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7801 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7861 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
     7921 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     7981 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     8041 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgtt
//
