LOCUS       sequence-430733-        6647 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 6647)
  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 6647)
  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: SSTR2-DuET
FEATURES             Location/Qualifiers
     source          1..6647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(587..606)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(588..606)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(651..854)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(680..700)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        855..1234
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1406..1425
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1500..1604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1605..1673,1674..2465)
                     /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(1823..1842)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2636..3224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3125..3144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3378..3395
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3512..3533
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3548..3553,3554..3571,3572..3578)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3586..3602
                     /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     3591..3613
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3610..3626
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3610..3626
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3675..3796
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3686..3705)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3740..3759
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3970..4764)
                     /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(4081..4100)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4691..4710
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4831..5160)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4845..4980
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4899..4918)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5145..5165
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5174..5602)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5284..5305)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5496..5515
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5648..5872
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5861..5878
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 ccgccacttg gctgacttta tcgggtggac gacggcaaga tagcggtcaa tactcataac
       61 agtcaaacag aaaattgaag tgaactggtt aattccgtct acagtcatta cgaccctaca
      121 tatagctttt ccgaaaggcc agtgaacgag tgccacctgc atcgccagga aaggcagtcc
      181 cagcataaac agttcatccg cgatggccag attgaggatg taaatgttgg taatagtttt
      241 catctttgca taccgcagga tgacgtaaat gacgagggta tttccgcaca atccgatgat
      301 gcacaccaca aagtaaatga aagtcaaaac ggcattgcta gtcaggtcat aatatggttc
      361 ggtctgattg ctcgtgttcg ttgacacgac tgaaccattc aaatcgaaag gtatgctcag
      421 ccaggtgtgg gaaccgttca gaggctcatc ggccatgtcc atatcgatgc tggcgtcatc
      481 atcgtccttg tagtcggcga ataccaggca gaagatgtag ctcagggcga tgatcgtctt
      541 catggtggcg ctaagcttaa gtttaaacgc tagccagctt gggtctccct atagtgagtc
      601 gtattaattt cgataagcca gtaagcagtg ggttctctag ttagccagag agctctgctt
      661 atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta
      721 cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg
      781 gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc
      841 caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga
      901 aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
      961 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
     1021 cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca
     1081 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
     1141 accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt
     1201 gattactatt aataactagt caataatcaa tgtcaacgcg tatatctggc ccgtacatcg
     1261 cgaagcagcg caaaacgcct aaccctaagc agattcttca tgcaaatgtc ggtcaagcct
     1321 tgccttgttg tagcttaaat tttgctcgcg cactactcag cgacctccaa cacacaagca
     1381 gggagcagat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     1441 aggggatcgg gagatctccc gatccgtcga cgtcaggtgg cacttttcgg ggaaatgtgc
     1501 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     1561 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     1621 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     1681 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     1741 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     1801 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     1861 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     1921 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     1981 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2041 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2101 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     2161 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     2221 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2281 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     2341 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     2401 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     2461 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     2521 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     2581 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2641 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2701 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2761 gagcgcagat accaaatacc gttcttctag tgtagccgta gttaggccac cacttcaaga
     2821 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2881 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2941 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3001 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3061 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3121 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3181 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3241 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3301 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3361 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     3421 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     3481 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     3541 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     3601 aatttcacac aggaaacagc tatgaccatg attacgccaa gctctagcta gaggtcgacg
     3661 gtatacagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     3721 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     3781 ctgcaataaa caagttgggg tgggcgaaga actccagcat gagatccccg cgctggagga
     3841 tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag aaggcggcgg
     3901 tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca
     3961 gagtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg
     4021 agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc
     4081 aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca
     4141 gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc
     4201 atgggtcacg acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc
     4261 ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc
     4321 catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc
     4381 cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg
     4441 agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct
     4501 tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca
     4561 cgatagccgc gctgcctcgt cctgcagttc attcagggca ccggacaggt cggtcttgac
     4621 aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat
     4681 tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc
     4741 gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagatcc
     4801 gaaaatggat atacaagctc ccgggagctt tttgcaaaag cctaggcctc caaaaaagcc
     4861 tcctcactac ttctggaata gctcagaggc agaggcggcc tcggcctctg cataaataaa
     4921 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat
     4981 gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact
     5041 tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat
     5101 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac
     5161 attccacaga attaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     5221 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     5281 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     5341 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     5401 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     5461 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     5521 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     5581 aatgcgccgc tacagggcgc gtggggatac cccctagagc cccagctggt tctttccgcc
     5641 tcagaagcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc
     5701 cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact cagacaatgc
     5761 gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc cagggtcaag
     5821 gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac agtcgaggct
     5881 gatcagcggg tttaaacggg ccctctagac tcgagttagg caggggccac ctgagaagtc
     5941 tcggtcttag acacggtggt gctggcctca tcgaattcac cggtgcgtcc accggtatcg
     6001 atgatggagg tctgcaggtc cccattgagg agggtccgtt gggtctcggt ggtctcatta
     6061 agtctacttt tgtcctgctt actgtcggat ctctcgccgt catcagttcc actcacctta
     6121 accaggcaca aaacgttttg aaagcttttc ttgaagttgt cgctaagaaa ggcgtacaga
     6181 atgggattgg cgcaactgtt ggcgtaggtc agaacgacca cgaaatcaaa cataccttta
     6241 agtgcagggg ttgggcttat ggccatagaa actgaactga cattgaaaat gtagaacggc
     6301 aaccaacaaa aaataaatac agccaccacg atagacacca ttctggtcac ttttttttca
     6361 gacttctttc gttttgaact tcccacccgt atcccgctgc tttttacctt aataattata
     6421 aacaggtaac acagacatat aattgtgagt ggtaccagga agccgagaat gaatgtatag
     6481 ataatgaacc ccgtgtacca tgctccactc tcgccgggcc aattgattgt gcaactagat
     6541 cggccccatt gatttgacct aaggcctgca tagatcataa ttggcaggat aaccagcagg
     6601 ctaacccccc acacagccat tgtgatcatt ttagcggttc ttggtct
//
