LOCUS       Exported                8111 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    OPRM1-Tango
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8111)
  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 8111)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8111
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2672..3673
                     /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(2739..2758)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3705..3722)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3711..3935
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3981..4409
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4068..4087)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4278..4299
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4418..4438)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4423..4752
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4603..4738
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4665..4684
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4819..5613
                     /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(4873..4892)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5483..5502
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5787..5908
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5824..5843)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5878..5897
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5957..5973)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5957..5973)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5970..5992)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5981..5997
                     /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(6005..6035)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6050..6071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6188..6205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6359..6944)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6439..6458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7115..7975)
                     /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     7738..7757
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7976..8080)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc
      961 agatatccag cacagtggcg gccgcgccac catgaagacg atcatcgccc tgagctacat
     1021 cttctgcctg gtattcgccg actacaagga cgatgatgac gccagcatcg atatgtgcct
     1081 tcatcggagg gtgccctccg aggagacata cagtctcgac cgcttcgctc aaaacccgcc
     1141 tttgttccct cccccaagcc tgccagcctc agaatccaga atggcgcatg caccgctgct
     1201 tcagcgatgc ggagcagcac gaaccggatt ctgcaagaaa cagcaggagc tttggcagag
     1261 gagaaaggag gccgctgagg ccctgggaac aagaaaggtg tccgtgctgc tggcaactag
     1321 ccacagcggt gcacggcccg ctgtgagcac catggactct tccgctgctc ccaccaatgc
     1381 ctcaaattgt acagacgcct tggcctatag ctcttgctct cccgcaccat ctccgggaag
     1441 ctgggttaac ctcagtcatc tcgacgggaa cctgtccgac ccttgtggcc ctaataggac
     1501 cgacctcggt ggtcgagata gcctgtgccc ccctactgga agccccagca tgatcacagc
     1561 tatcacaata atggctctct atagtatcgt ctgcgtcgtg ggactctttg gcaattttct
     1621 cgtcatgtac gttatcgtca gatacacaaa gatgaaaacc gcaaccaata tttacatctt
     1681 taatctggcc ctggcggatg cccttgcaac cagtacactg ccgtttcaat ccgtgaatta
     1741 tctgatgggc acatggccct tcggaaccat tctgtgcaaa atcgtaatct caattgatta
     1801 ttataacatg ttcacctcca tttttacctt gtgtacaatg tcagtagacc gctatatcgc
     1861 cgtatgccac cctgtcaaag ctcttgattt caggacccct aggaatgcca agatcatcaa
     1921 tgtgtgcaat tggattctgt ctagcgctat cggcttgccg gtgatgttca tggccactac
     1981 caagtacaga caaggttcaa tcgactgtac cctcacattt tcacatccca cgtggtattg
     2041 ggagaacttg ctgaagatct gtgtgttcat cttcgccttc atcatgccag tactgataat
     2101 caccgtttgt tatggtctta tgatcctgcg cctgaagagc gtgcgcatgc tcagcggctc
     2161 caaagaaaag gaccggaacc ttcggcgaat cacacggatg gtcctggtgg ttgtggctgt
     2221 gttcattgtt tgctggaccc ctattcatat ttatgttata attaaggccc tggtgaccat
     2281 cccggaaacc acattccaga ccgtttcctg gcacttttgt atagcattgg gatataccaa
     2341 cagttgcctt aatcccgttc tgtacgcgtt tctggacgag aatttcaagc gatgcttcag
     2401 ggagttctgc attcccacaa gctctaacat tgaacagcag aattctacac gaatacgaca
     2461 gaataccaga gatcatccta gcactgccaa tactgtcgac cggaccaatc accagcttga
     2521 gaaccttgag gcagaaaccg cccctctccc aatcgatacc ggtggacgca ccccacccag
     2581 cctgggtccc caagatgagt cctgcaccac cgccagctcc tccctggcca aggacacttc
     2641 atcgaccggt gagaacctgt acttccagct aagattagat aaaagtaaag tgattaacag
     2701 cgcattagag ctgcttaatg aggtcggaat cgaaggttta acaacccgta aactcgccca
     2761 gaagctaggt gtagagcagc ctacattgta ttggcatgta aaaaataagc gggctttgct
     2821 cgacgcctta gccattgaga tgttagatag gcaccatact cacttttgcc ctttagaagg
     2881 ggaaagctgg caagattttt tacgtaataa cgctaaaagt tttagatgtg ctttactaag
     2941 tcatcgcgat ggagcaaaag tacatttagg tacacggcct acagaaaaac agtatgaaac
     3001 tctcgaaaat caattagcct ttttatgcca acaaggtttt tcactagaga atgcattata
     3061 tgcactcagc gctgtggggc attttacttt aggttgcgta ttggaagatc aagagcatca
     3121 agtcgctaaa gaagaaaggg aaacacctac tactgatagt atgccgccat tattacgaca
     3181 agctatcgaa ttatttgatc accaaggtgc agagccagcc ttcttattcg gccttgaatt
     3241 gatcatatgc ggattagaaa aacaacttaa atgtgaaagt gggtccgcgt acagccgcgc
     3301 gcgtacgaaa aacaattacg ggtctaccat cgagggcctg ctcgatctcc cggacgacga
     3361 cgcccccgaa gaggcggggc tggcggctcc gcgcctgtcc tttctccccg cgggacacac
     3421 gcgcagactg tcgacggccc ccccgaccga tgtcagcctg ggggacgagc tccacttaga
     3481 cggcgaggac gtggcgatgg cgcatgccga cgcgctagac gatttcgatc tggacatgtt
     3541 gggggacggg gattccccgg gtccgggatt taccccccac gactccgccc cctacggcgc
     3601 tctggatatg gccgacttcg agtttgagca gatgtttacc gatgcccttg gaattgacga
     3661 gtacggtggg tagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     3721 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3781 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3841 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3901 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3961 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4021 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     4081 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4141 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     4201 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4261 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     4321 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     4381 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     4441 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4501 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4561 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     4621 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4681 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4741 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     4801 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     4861 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     4921 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     4981 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     5041 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     5101 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     5161 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     5221 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     5281 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     5341 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     5401 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     5461 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     5521 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     5581 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     5641 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     5701 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     5761 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     5821 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5881 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     5941 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     6001 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     6061 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6121 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     6181 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     6241 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     6301 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     6361 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     6421 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     6481 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6541 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6601 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6661 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6721 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6781 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6841 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggttttt
     6901 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6961 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     7021 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     7081 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7141 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7201 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7261 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7321 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7381 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7441 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7501 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7561 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7621 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7681 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7741 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7801 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7861 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7921 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7981 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     8041 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     8101 cacctgacgt c
//
