LOCUS       Exported                8018 bp ds-DNA     circular SYN 12-NOV-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    CALCRL-Tango
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8018)
  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 8018)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8018
                     /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             2342..3343
                     /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(2409..2428)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3375..3392)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3381..3605
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3651..4079
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3738..3757)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3948..3969
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4088..4108)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4093..4422
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4273..4408
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4335..4354
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4489..5283
                     /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(4543..4562)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5153..5172
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5457..5578
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5494..5513)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5548..5567
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5627..5643)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5627..5643)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5640..5662)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5651..5667
                     /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(5675..5705)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5720..5741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5858..5875)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6029..6617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6109..6128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6788..7648)
                     /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     7411..7430
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7649..7753)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7828..7847)
                     /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 ggagaaaaag tgcacgctct acttccttgt gctgctgccc ttttttatga tcctcgtgac
      901 tgccgagctg gaagagagtc ccgaggactc aatccagctg ggagtcacgc ggaacaaaat
      961 catgacagca caatacgagt gttaccagaa gataatgcag gaccctatcc agcaagcaga
     1021 aggggtctat tgcaaccgca cgtgggacgg atggttgtgc tggaatgacg tcgccgccgg
     1081 cacagagtct atgcagctct gtccagatta tttccaggat tttgacccat ccgaaaaagt
     1141 aactaagatt tgtgatcagg atggcaattg gtttaggcac cccgcatcaa accgcacatg
     1201 gacaaattac acacagtgca atgtgaatac ccacgaaaaa gtcaaaactg ccctgaacct
     1261 gttttacctc acaatcatag gccacggtct ctcaatagcc tccctcctca tttctctggg
     1321 catattcttc tattttaaaa gcttgagctg ccagaggatt acactccata aaaacctgtt
     1381 cttctcattc gtttgcaata gcgtagtgac tatcatacat ttgacagcgg tcgccaataa
     1441 ccaggcgctg gtcgccacta atcccgtgtc ttgcaaagtc tcccaattca ttcacttgta
     1501 cctgatgggc tgcaactact tctggatgct ctgtgagggt atatatcttc atacattgat
     1561 agtggtggct gtgttcgctg agaagcagca tcttatgtgg tactactttt tggggtgggg
     1621 gttccctttg attccggcct gtattcatgc gatcgctagg tcactgtact acaacgataa
     1681 ctgttggatc tcatctgaca cgcacctgct ctacattatt catggcccaa tctgcgccgc
     1741 tctgctggtc aatctgttct tccttctgaa catagtgaga gtgctgatca caaaattgaa
     1801 ggtcactcat caggcagaaa gcaacctgta catgaaggcc gtcagagcca ctttgatctt
     1861 ggtacctctg ctgggtatcg aattcgtgct gattccatgg aggcctgaag gaaaaatcgc
     1921 agaggaagtg tacgattaca ttatgcacat tctcatgcat ttccaggggc ttctggtgag
     1981 caccattttc tgctttttta acggtgaagt gcaggccata ctccggcgga attggaatca
     2041 atacaagata cagtttggca atagcttttc aaatagtgaa gccctcaggt ccgccagtta
     2101 cacagtaagt accataagcg atgggccggg ctactctcat gactgcccat ctgaacatct
     2161 taacgggaaa tctattcacg atattgagaa cgtcctgctg aagccagaaa atctgtacaa
     2221 catcgatacc ggtggacgca ccccacccag cctgggtccc caagatgagt cctgcaccac
     2281 cgccagctcc tccctggcca aggacacttc atcgaccggt gagaacctgt acttccagct
     2341 aagattagat aaaagtaaag tgattaacag cgcattagag ctgcttaatg aggtcggaat
     2401 cgaaggttta acaacccgta aactcgccca gaagctaggt gtagagcagc ctacattgta
     2461 ttggcatgta aaaaataagc gggctttgct cgacgcctta gccattgaga tgttagatag
     2521 gcaccatact cacttttgcc ctttagaagg ggaaagctgg caagattttt tacgtaataa
     2581 cgctaaaagt tttagatgtg ctttactaag tcatcgcgat ggagcaaaag tacatttagg
     2641 tacacggcct acagaaaaac agtatgaaac tctcgaaaat caattagcct ttttatgcca
     2701 acaaggtttt tcactagaga atgcattata tgcactcagc gctgtggggc attttacttt
     2761 aggttgcgta ttggaagatc aagagcatca agtcgctaaa gaagaaaggg aaacacctac
     2821 tactgatagt atgccgccat tattacgaca agctatcgaa ttatttgatc accaaggtgc
     2881 agagccagcc ttcttattcg gccttgaatt gatcatatgc ggattagaaa aacaacttaa
     2941 atgtgaaagt gggtccgcgt acagccgcgc gcgtacgaaa aacaattacg ggtctaccat
     3001 cgagggcctg ctcgatctcc cggacgacga cgcccccgaa gaggcggggc tggcggctcc
     3061 gcgcctgtcc tttctccccg cgggacacac gcgcagactg tcgacggccc ccccgaccga
     3121 tgtcagcctg ggggacgagc tccacttaga cggcgaggac gtggcgatgg cgcatgccga
     3181 cgcgctagac gatttcgatc tggacatgtt gggggacggg gattccccgg gtccgggatt
     3241 taccccccac gactccgccc cctacggcgc tctggatatg gccgacttcg agtttgagca
     3301 gatgtttacc gatgcccttg gaattgacga gtacggtggg tagtctagag ggcccgttta
     3361 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3421 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3481 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3541 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3601 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     3661 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3721 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3781 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3841 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3901 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3961 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4021 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     4081 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     4141 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     4201 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4261 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4321 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     4381 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     4441 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     4501 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4561 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4621 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     4681 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4741 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     4801 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4861 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     4921 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     4981 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     5041 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     5101 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     5161 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     5221 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     5281 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     5341 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     5401 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     5461 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5521 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5581 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5641 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5701 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5761 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5821 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5881 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5941 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6001 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6061 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6121 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6181 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6241 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6301 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6361 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6421 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6481 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6541 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6601 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6661 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6721 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6781 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6841 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6901 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6961 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7021 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7081 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7141 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7201 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7261 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7321 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7381 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7441 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7501 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7561 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7621 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7681 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7741 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7801 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     7861 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     7921 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     7981 tgcgctgctt cgcgatgtac gggccagata tacgcgtt
//
