LOCUS       Exported                8057 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    HTR2B-Tango
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8057)
  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 8057)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8057
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(6..25)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        197..576
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        577..780
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     731..751
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     825..844
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        825..843
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2598..3599
                     /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(2665..2684)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3631..3648)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3637..3861
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3907..4335
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3994..4013)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4204..4225
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4344..4364)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4509..4643
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4570..4589
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4724..5518
                     /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(4778..4797)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5388..5407
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5692..5813
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5729..5748)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5783..5802
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5862..5878)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5862..5878)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5875..5897)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5886..5902
                     /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(5910..5940)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5955..5976
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6093..6110)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6264..6852)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6344..6363)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7023..7883)
                     /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     7646..7665
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7884..7988)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
       61 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
      121 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
      181 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
      241 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      301 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      361 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      421 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      481 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      541 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      601 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      661 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      721 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
      781 ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat
      841 agggagaccc aagctggcta gcgtttaaac ttaagcttgg taccgagctc ggatccacta
      901 gtccagtgtg gtggaattct gcagatatcc agcacagtgg cggccgcgcc accatgaaga
      961 cgatcatcgc cctgagctac atcttctgcc tggtattcgc cgactacaag gacgatgatg
     1021 acgccagcat cgatatggcc ctgtcttatc gggtgagcga gcttcaatct accatcccag
     1081 agcacatttt gcagagcact ttcgtacatg tcattagttc caattggtct gggctccaga
     1141 ctgaatctat cccagaggag atgaagcaaa tcgtggagga gcagggtaac aaactgcact
     1201 gggcagccct gctcatactg atggtgatca tcccgacaat tggaggcaat actctcgtga
     1261 tccttgcggt tagcctggag aagaaactgc agtacgcaac caattacttt ctcatgagcc
     1321 tcgccgtcgc cgatctcctg gtggggttgt ttgttatgcc tatcgcactt ctcaccatca
     1381 tgttcgaagc tatgtggccc ctccctcttg tgctgtgccc tgcatggttg ttccttgatg
     1441 tgctgttcag cacagcaagc atcatgcatt tgtgtgccat ctccgtggac cgatacattg
     1501 ctatcaaaaa acccattcag gccaatcaat acaacagccg ggccaccgcc tttatcaaga
     1561 taaccgttgt gtggcttatc agcatcggta tcgccatccc cgtgccgatc aaggggatcg
     1621 agacggatgt ggacaatcca aacaatatca catgcgtact caccaaggag aggttcggag
     1681 actttatgct gttcgggagc cttgccgcat tctttacacc tcttgccatc atgattgtga
     1741 catattttct gaccatacat gcgctccaga aaaaagccta tctggttaag aacaaaccgc
     1801 cccaacggct gacttggctg acagtgtcca ccgtcttcca gcgcgacgag accccgtgta
     1861 gttcccctga aaaagtggcc atgctcgacg gttcccgaaa ggacaaagcc cttcctaatt
     1921 ctggcgatga gacacttatg cgccgaacct ctacgattgg caaaaagtct gttcagacaa
     1981 tctccaacga acagcgggcg tccaaagttc tgggtattgt gttcttcctg tttctgctga
     2041 tgtggtgccc tttcttcatc acaaacataa ccctggtgct ttgcgactct tgcaaccaga
     2101 ccacattgca gatgctgctc gaaatattcg tgtggatcgg ctacgtgagc agcggagtga
     2161 acccccttgt gtacactctg ttcaataaga ctttccgcga cgcctttggg agatatatca
     2221 cctgcaacta cagagccaca aagtccgtga agaccttgcg gaagcggtca tccaaaatct
     2281 attttagaaa tcctatggca gaaaattcca agttctttaa aaagcacgga atacggaacg
     2341 gaataaaccc cgcgatgtac cagtctccaa tgaggcttcg gtcctctaca atacagtcca
     2401 gcagcatcat cctgcttgat actctgctgc tcactgagaa tgaaggagat aaaaccgagg
     2461 agcaggtctc ctatgtgatc gataccggtg gacgcacccc acccagcctg ggtccccaag
     2521 atgagtcctg caccaccgcc agctcctccc tggccaagga cacttcatcg accggtgaga
     2581 acctgtactt ccagctaaga ttagataaaa gtaaagtgat taacagcgca ttagagctgc
     2641 ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag ctaggtgtag
     2701 agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac gccttagcca
     2761 ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa agctggcaag
     2821 attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat cgcgatggag
     2881 caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc gaaaatcaat
     2941 tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca ctcagcgctg
     3001 tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag
     3061 aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct atcgaattat
     3121 ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc atatgcggat
     3181 tagaaaaaca acttaaatgt gaaagtgggt ccgcgtacag ccgcgcgcgt acgaaaaaca
     3241 attacgggtc taccatcgag ggcctgctcg atctcccgga cgacgacgcc cccgaagagg
     3301 cggggctggc ggctccgcgc ctgtcctttc tccccgcggg acacacgcgc agactgtcga
     3361 cggccccccc gaccgatgtc agcctggggg acgagctcca cttagacggc gaggacgtgg
     3421 cgatggcgca tgccgacgcg ctagacgatt tcgatctgga catgttgggg gacggggatt
     3481 ccccgggtcc gggatttacc ccccacgact ccgcccccta cggcgctctg gatatggccg
     3541 acttcgagtt tgagcagatg tttaccgatg cccttggaat tgacgagtac ggtgggtagt
     3601 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     3661 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     3721 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3781 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     3841 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     3901 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3961 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4021 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4081 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4141 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4201 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4261 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4321 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     4381 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     4441 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4501 aaccatagtc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4561 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     4621 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     4681 cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     4741 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     4801 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     4861 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     4921 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     4981 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     5041 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     5101 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     5161 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     5221 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     5281 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     5341 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     5401 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     5461 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     5521 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     5581 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     5641 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     5701 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     5761 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     5821 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5881 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5941 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     6001 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     6061 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     6121 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     6181 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6241 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6301 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6361 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6421 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6481 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6541 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6601 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6661 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6721 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6781 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6841 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6901 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6961 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     7021 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     7081 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     7141 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     7201 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     7261 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     7321 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7381 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7441 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7501 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7561 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7621 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7681 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7741 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7801 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7861 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7921 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7981 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
     8041 atcccctatg gtgcact
//
