LOCUS       Exported                8048 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    HTR2A-Tango
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8048)
  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 8048)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8048
                     /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             2606..3607
                     /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(2673..2692)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3639..3656)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3645..3869
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3915..4343
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4002..4021)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4212..4233
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4352..4372)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4357..4686
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4537..4672
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4599..4618
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4753..5547
                     /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(4807..4826)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5417..5436
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5721..5842
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5758..5777)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5812..5831
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5891..5907)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5891..5907)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5904..5926)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5915..5931
                     /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(5939..5969)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5984..6005
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6122..6139)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6293..6881)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6373..6392)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7052..7912)
                     /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     7675..7694
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7913..8017)
                     /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 atatggatat
     1081 cttgtgcgag gagaatacct cactgtccag cacaactaac tctctcatgc agctcaatga
     1141 tgatacccga ctttactcca acgacttcaa cagtggtgag gctaatacat cagacgcctt
     1201 caattggaca gtagacagtg aaaaccggac caatctgtcc tgtgagggat gcctgtcccc
     1261 aagttgtttg tctctcctgc atttgcagga aaagaactgg tctgcgctgc tcaccgccgt
     1321 ggttatcata ctcactatag ctggcaatat tctcgtaatc atggccgtgt ccctggaaaa
     1381 aaagctgcag aacgccacga actacttcct gatgtctctg gcgattgcgg atatgctgct
     1441 tgggtttctg gtgatgcctg tatccatgtt gactatactg tacgggtacc gctggccact
     1501 cccatccaag ttgtgtgccg tctggatcta tctggacgtt ctctttagta ccgcaagcat
     1561 aatgcacctt tgtgctatta gcctggaccg ctacgtcgct attcaaaacc ccatccacca
     1621 cagcagattc aacagccgaa ctaaagcctt tctgaaaatc atcgcggtct ggaccatttc
     1681 agttggcatc agcatgccga tacctgtatt tggccttcag gatgactcta aggtcttcaa
     1741 agagggcagt tgccttctgg ctgatgacaa tttcgtactt ataggaagct tcgtgtcctt
     1801 cttcattcct cttaccatta tggtgatcac ttacttcctg acaattaagt cccttcaaaa
     1861 ggaagcgacc ctttgcgtta gcgatttggg cacccgggcc aagcttgcaa gttttagctt
     1921 cctgcctcaa tcctccctga gcagcgagaa actgttccag cggagcatac accgagagcc
     1981 cgggtcttat actggccggc gcaccatgca aagcatcagc aacgagcaga aggcttgcaa
     2041 ggtcctgggc attgtgtttt tcctgtttgt tgtgatgtgg tgtccattct ttataacgaa
     2101 catcatggcg gttatctgca aagagtcctg caacgaggat gttatcggag ctttgttgaa
     2161 cgtgtttgta tggatcggat atctgtcctc agctgtgaat ccattggtct acaccctttt
     2221 taacaaaacc tatcggtccg ctttttcccg gtacatccag tgtcagtaca aagagaataa
     2281 gaagccactc cagcttatac tggtcaatac tatacccgca ctggcatata agagctccca
     2341 actgcagatg gggcagaaaa aaaactctaa acaagatgcc aagaccacag acaacgactg
     2401 cagtatggtg gccctgggca aacagcacag tgaagaagcc tccaaagaca atagtgacgg
     2461 cgttaacgag aaagtctcct gcgttatcga taccggtgga cgcaccccac ccagcctggg
     2521 tccccaagat gagtcctgca ccaccgccag ctcctccctg gccaaggaca cttcatcgac
     2581 cggtgagaac ctgtacttcc agctaagatt agataaaagt aaagtgatta acagcgcatt
     2641 agagctgctt aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg cccagaagct
     2701 aggtgtagag cagcctacat tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc
     2761 cttagccatt gagatgttag ataggcacca tactcacttt tgccctttag aaggggaaag
     2821 ctggcaagat tttttacgta ataacgctaa aagttttaga tgtgctttac taagtcatcg
     2881 cgatggagca aaagtacatt taggtacacg gcctacagaa aaacagtatg aaactctcga
     2941 aaatcaatta gcctttttat gccaacaagg tttttcacta gagaatgcat tatatgcact
     3001 cagcgctgtg gggcatttta ctttaggttg cgtattggaa gatcaagagc atcaagtcgc
     3061 taaagaagaa agggaaacac ctactactga tagtatgccg ccattattac gacaagctat
     3121 cgaattattt gatcaccaag gtgcagagcc agccttctta ttcggccttg aattgatcat
     3181 atgcggatta gaaaaacaac ttaaatgtga aagtgggtcc gcgtacagcc gcgcgcgtac
     3241 gaaaaacaat tacgggtcta ccatcgaggg cctgctcgat ctcccggacg acgacgcccc
     3301 cgaagaggcg gggctggcgg ctccgcgcct gtcctttctc cccgcgggac acacgcgcag
     3361 actgtcgacg gcccccccga ccgatgtcag cctgggggac gagctccact tagacggcga
     3421 ggacgtggcg atggcgcatg ccgacgcgct agacgatttc gatctggaca tgttggggga
     3481 cggggattcc ccgggtccgg gatttacccc ccacgactcc gccccctacg gcgctctgga
     3541 tatggccgac ttcgagtttg agcagatgtt taccgatgcc cttggaattg acgagtacgg
     3601 tgggtagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     3661 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3721 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3781 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3841 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3901 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3961 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4021 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4081 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     4141 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4201 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     4261 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     4321 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     4381 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4441 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4501 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4561 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4621 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4681 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     4741 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4801 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4861 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4921 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4981 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     5041 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     5101 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     5161 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     5221 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     5281 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     5341 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     5401 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     5461 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     5521 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     5581 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5641 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5701 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     5761 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5821 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5881 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5941 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     6001 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     6061 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     6121 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     6181 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6241 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6301 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6361 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6421 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6481 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6541 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6601 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6661 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6721 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6781 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6841 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6901 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6961 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     7021 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     7081 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     7141 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     7201 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7261 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7321 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7381 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7441 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7501 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7561 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7621 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7681 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7741 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7801 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7861 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7921 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7981 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     8041 ctgacgtc
//
