LOCUS       sequence-430788-        6578 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The DuET construct encodes the named human GPCR engineered with a 
            cleaved hemagglutinin signal peptide, a 5' FLAG and a 3' 1D4 epitope
            tag optimized for expression in mammalian cells and proteomics 
            studies..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6578)
  AUTHORS   Dahl L, Kotliar IB, Bendes A, Dodig-Crnkovic T, Fromm S, Elofsson A,
            Uhlen M, Sakmar TP, Schwenk JM
  TITLE     Multiplexed selectivity screening of anti-GPCR antibodies.
  JOURNAL   Sci Adv. 2023 May 3;9(18):eadf9297. doi: 10.1126/sciadv.adf9297. 
            Epub 2023 May 3.
  PUBMED    37134173
REFERENCE   2  (bases 1 to 6578)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2023-05-3"; volume: "9(18):eadf9297. doi"; pages: " 
            10.1126/sciadv.adf9297. Epub 2023 May 3"
COMMENT     Sequence Label: CYSLTR2-DuET
FEATURES             Location/Qualifiers
     source          1..6578
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     221..240
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        315..419
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(420..488,489..1280)
                     /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     complement(638..657)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1451..2039
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1940..1959
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2193..2210
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2327..2348
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2363..2368,2369..2386,2387..2393)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2401..2417
                     /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)."
     primer_bind     2406..2428
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2425..2441
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2425..2441
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2490..2611
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2501..2520)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2555..2574
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2785..3579)
                     /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(2896..2915)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3506..3525
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3646..3975)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3660..3795
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3714..3733)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3960..3980
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3989..4417)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4099..4120)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4311..4330
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    4463..4687
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4676..4693
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5980..5999)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5981..5999)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6044..6247)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6073..6093)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        join(6248..6578,1..49)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca acgcgtatat
       61 ctggcccgta catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt cttcatgcaa
      121 atgtcggtca agccttgcct tgttgtagct taaattttgc tcgcgcacta ctcagcgacc
      181 tccaacacac aagcagggag cagatactgg cttaactatg cggcatcaga gcagattgta
      241 ctgagagtgc accatagggg atcgggagat ctcccgatcc gtcgacgtca ggtggcactt
      301 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
      361 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
      421 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
      481 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
      541 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
      601 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
      661 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
      721 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
      781 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
      841 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
      901 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
      961 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     1021 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     1081 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     1141 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     1201 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     1261 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     1321 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     1381 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     1441 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     1501 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     1561 taactggctt cagcagagcg cagataccaa ataccgttct tctagtgtag ccgtagttag
     1621 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     1681 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     1741 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     1801 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     1861 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     1921 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     1981 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2041 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2101 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2161 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2221 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     2281 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     2341 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     2401 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct
     2461 agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac
     2521 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     2581 tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat
     2641 ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt
     2701 catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg
     2761 tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
     2821 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
     2881 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
     2941 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
     3001 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag
     3061 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
     3121 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
     3181 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     3241 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     3301 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     3361 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
     3421 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     3481 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     3541 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     3601 ctcttgatca gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag
     3661 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc
     3721 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     3781 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     3841 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga
     3901 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     3961 ccctaactga cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca
     4021 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     4081 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     4141 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     4201 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     4261 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     4321 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     4381 acacccgccg cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag
     4441 ctggttcttt ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg
     4501 tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac
     4561 ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca
     4621 ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa
     4681 ggcacagtcg aggctgatca gcgggtttaa acgggccctc tagactcgag ttaggcaggg
     4741 gccacctgag aagtctcggt cttagacacg gtggtgctgg cctcatcgaa ttcaccggtg
     4801 cgtccaccgg tatcgatcac cctcgtctcc ttgcgcagcc acacagaaac ggggaacacg
     4861 cacttggttt tagccttctg cggatgacct ttcctcagtg cggatttcag ccggtccttg
     4921 aagttctctc cggcaaagta atacaggagg ggattgaaac aggcattagc cgccgccagc
     4981 gccaaggtaa ttaccagagc cttgtgcagc ctatccttac acagacccac cttccatgtt
     5041 gtcagatgca cagtgcgcag cgtgtgatat ggaaggaagc acaggaagaa aatgataagc
     5101 gtaataatta tggtagtcag ggctttcctg tgactcactc tcagtccgct ctcgggcact
     5161 tccactttga gcaatactcg aattatcagg aggtaacaga tgctgagtgt gaagaaaggc
     5221 agcagacacc ccaccacaag agcaatgtag ttcattgttt gcagtttagc gattttatac
     5281 aggttcagtt ccaggcagct agtgacactt ccattttgct cagaaccgct gtccagaagc
     5341 ataatggaag aggccattat cagtatccat atgatgccac acaagatcca ggcgcttcta
     5401 atggatgtga catgcaacag tcggaatggg tgcaccatgg ccaggaacct gaccacagac
     5461 agcacggtca ggaaatagat actgctgtac atgttcacgt acaggctgta actcataatt
     5521 ctacaagcga ggtctccaaa aatccaattg ctacctctaa gatagtaatc tgccctaaag
     5581 ggcaacgtgc tgataaagag caagtcagag atcgccagat tcagcataaa cacattcaca
     5641 gatgtggact ttttgtaggg ctggaggaag acgtaaatag aaagtccatt gccgagaact
     5701 ccccaaaaaa aaataatcaa gtacacgata gggaagaatt ctcgtttaaa gttctcgatg
     5761 gtgcaattcc gagaattatt atttgagaag gtgccattgg gttccatttc gctcacgctg
     5821 atagagggtt gcagagacat aaattttctt tccatatcga tgctggcgtc atcatcgtcc
     5881 ttgtagtcgg cgaataccag gcagaagatg tagctcaggg cgatgatcgt cttcatggtg
     5941 gcgctaagct taagtttaaa cgctagccag cttgggtctc cctatagtga gtcgtattaa
     6001 tttcgataag ccagtaagca gtgggttctc tagttagcca gagagctctg cttatataga
     6061 cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt
     6121 ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca atggggtgga
     6181 gacttggaaa tccccgtgag tcaaaccgct atccacgccc attgatgtac tgccaaaacc
     6241 gcatcaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc
     6301 ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag
     6361 ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat
     6421 actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca
     6481 ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa
     6541 gttatgtaac gcggaactcc atatatgggc tatgaact
//
