LOCUS       Exported                7631 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    MC4R-Tango
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7631)
  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 7631)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7631
                     /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             1955..2956
                     /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(2022..2041)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(2988..3005)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2994..3218
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3264..3692
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3351..3370)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3561..3582
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3701..3721)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3706..4035
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3886..4021
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3948..3967
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4102..4896
                     /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(4156..4175)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4766..4785
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5070..5191
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5107..5126)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5161..5180
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5240..5256)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5240..5256)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5253..5275)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5264..5280
                     /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(5288..5318)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5333..5354
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5471..5488)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5642..6230)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5722..5741)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6401..7261)
                     /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     7024..7043
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7262..7366)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7441..7460)
                     /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 ggtgaatagc acgcaccgcg gaatgcacac gagccttcat ctgtggaaca gaagctctta
      901 tcggctccat tctaatgcct cagagtcact cgggaagggg tactccgacg gaggttgcta
      961 tgagcagctg ttcgtaagcc ccgaagtttt cgtgaccctg ggagtgattt cactcctgga
     1021 aaacatactg gtgatagtgg ctatcgctaa aaataaaaac ctccacagtc ctatgtactt
     1081 cttcatctgc agtttggctg tcgccgatat gctcgtgagt gtttcaaatg ggagtgagac
     1141 catagtgatc actttgctga acagcacaga taccgatgct caaagcttta ctgtaaatat
     1201 agataacgtg attgatagcg tcatctgttc ttctctgctt gcgtctattt gcagtttgtt
     1261 gagcattgca gtcgaccgct acttcactat tttctacgcc ctgcagtatc ataacataat
     1321 gacagtgaag cgcgtgggta tcattatctc ctgtatctgg gcagcttgta ctgttagcgg
     1381 cattctgttt ataatttatt ccgatagtag cgccgtcatt atctgtttga ttacaatgtt
     1441 cttcactatg ctcgctctca tggccagtct gtatgtccat atgtttctga tggctagact
     1501 ccatatcaag cggatcgccg tcctgccggg aaccggagct attcgccaag gcgcgaatat
     1561 gaagggggcg attaccctta caatcctgat cggcgtgttt gtggtgtgtt gggcgccctt
     1621 cttcctgcac ctgattttct acatatcctg cccccagaat ccttactgcg tgtgcttcat
     1681 gtcacatttc aacctgtatc tgattctgat tatgtgcaac agcatcatag atcccctcat
     1741 ttatgcactg agaagtcaag agctgcggaa gaccttcaag gagatcatct gttgctaccc
     1801 gctcggcgga ctgtgtgatc tgtcctccag gtatatcgat accggtggac gcaccccacc
     1861 cagcctgggt ccccaagatg agtcctgcac caccgccagc tcctccctgg ccaaggacac
     1921 ttcatcgacc ggtgagaacc tgtacttcca gctaagatta gataaaagta aagtgattaa
     1981 cagcgcatta gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc gtaaactcgc
     2041 ccagaagcta ggtgtagagc agcctacatt gtattggcat gtaaaaaata agcgggcttt
     2101 gctcgacgcc ttagccattg agatgttaga taggcaccat actcactttt gccctttaga
     2161 aggggaaagc tggcaagatt ttttacgtaa taacgctaaa agttttagat gtgctttact
     2221 aagtcatcgc gatggagcaa aagtacattt aggtacacgg cctacagaaa aacagtatga
     2281 aactctcgaa aatcaattag cctttttatg ccaacaaggt ttttcactag agaatgcatt
     2341 atatgcactc agcgctgtgg ggcattttac tttaggttgc gtattggaag atcaagagca
     2401 tcaagtcgct aaagaagaaa gggaaacacc tactactgat agtatgccgc cattattacg
     2461 acaagctatc gaattatttg atcaccaagg tgcagagcca gccttcttat tcggccttga
     2521 attgatcata tgcggattag aaaaacaact taaatgtgaa agtgggtccg cgtacagccg
     2581 cgcgcgtacg aaaaacaatt acgggtctac catcgagggc ctgctcgatc tcccggacga
     2641 cgacgccccc gaagaggcgg ggctggcggc tccgcgcctg tcctttctcc ccgcgggaca
     2701 cacgcgcaga ctgtcgacgg cccccccgac cgatgtcagc ctgggggacg agctccactt
     2761 agacggcgag gacgtggcga tggcgcatgc cgacgcgcta gacgatttcg atctggacat
     2821 gttgggggac ggggattccc cgggtccggg atttaccccc cacgactccg ccccctacgg
     2881 cgctctggat atggccgact tcgagtttga gcagatgttt accgatgccc ttggaattga
     2941 cgagtacggt gggtagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     3001 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3061 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3121 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3181 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3241 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3301 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3361 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3421 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3481 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3541 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3601 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3661 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     3721 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3781 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3841 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3901 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3961 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4021 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     4081 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4141 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4201 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4261 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     4321 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4381 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4441 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4501 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4561 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4621 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4681 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4741 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4801 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4861 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4921 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     4981 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5041 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     5101 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5161 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5221 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5281 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5341 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5401 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5461 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5521 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5581 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5641 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5701 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5761 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5821 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5881 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5941 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6001 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6061 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6121 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6181 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6241 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6301 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6361 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6421 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6481 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6541 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6601 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6661 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6721 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6781 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6841 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6901 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6961 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7021 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7081 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7141 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7201 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7261 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7321 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7381 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     7441 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     7501 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     7561 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     7621 atatacgcgt t
//
