LOCUS       sequence-430751-        6680 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 6680)
  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 6680)
  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: APJ-DuET
FEATURES             Location/Qualifiers
     source          1..6680
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(83..671)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(163..182)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(842..1633,1634..1702))
                     /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     1465..1484
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1703..1807)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1882..1901)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2073..2452
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2453..2656
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2607..2627
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2701..2720
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2701..2719
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4109..4126)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4115..4339
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4385..4813
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4472..4491)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4682..4703
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4822..4842)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4827..5156
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5007..5142
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5069..5088
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5223..6017
                     /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(5277..5296)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5887..5906
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6191..6312
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6228..6247)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6282..6301
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6361..6377)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6361..6377)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6374..6396)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6385..6401
                     /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(join(6409..6415,6416..6433,6434..6439))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6454..6475
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6592..6609)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
       61 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
      121 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
      181 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
      241 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
      301 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
      361 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
      421 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
      481 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacggta tttggtatct
      541 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
      601 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
      661 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
      721 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
      781 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
      841 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
      901 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
      961 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     1021 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     1081 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     1141 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     1201 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     1261 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     1321 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     1381 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     1441 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     1501 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     1561 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     1621 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     1681 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     1741 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     1801 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     1861 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     1921 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     1981 aaggcaaggc ttgaccgaca tttgcatgaa gaatctgctt agggttaggc gttttgcgct
     2041 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
     2101 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     2161 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     2221 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     2281 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     2341 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     2401 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     2461 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     2521 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     2581 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     2641 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
     2701 taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa gcttagcgcc
     2761 accatgaaga cgatcatcgc cctgagctac atcttctgcc tggtattcgc cgactacaag
     2821 gacgatgatg acgccagcat cgatatggag gagggcggag actttgacaa ttattacggt
     2881 gccgataatc agtccgagtg cgagtacact gactggaaaa gctccggtgc actcatccct
     2941 gctatatata tgctggtgtt tctgttggga accactggaa acggcctcgt actgtggact
     3001 gtgtttagga gttcccggga aaaacggagg agcgccgata tttttattgc aagcctggcc
     3061 gtggctgacc ttacgtttgt ggtgaccctg ccactgtggg caacctacac atatcgcgat
     3121 tacgattggc ctttcggaac attcttctgc aaactgagtt cttatctcat cttcgttaat
     3181 atgtatgcat ctgtcttctg tctgacgggc ctcagttttg atcggtacct cgcgattgtt
     3241 cggcccgtag ctaatgccag actgaggctg agagtcagtg gggccgtcgc cacagcagtg
     3301 ctctgggtgt tggcggctct gcttgcgatg ccagtaatgg tactcaggac aaccggagac
     3361 ctggagaaca ctacaaaagt gcagtgttat atggattata gcatggtcgc taccgtctca
     3421 agcgagtggg cttgggaagt ggggttgggc gtgagcagca ctacagttgg gttcgtggta
     3481 cccttcacga ttatgctgac atgctacttc ttcattgccc agaccatcgc tggtcacttc
     3541 cggaaagaga ggattgaagg cctccggaag cggaggcggc tgctgagcat cattgttgtg
     3601 ctggtcgtga cctttgctct ctgttggatg ccgtatcatc tggtgaaaac actctacatg
     3661 ctcggatccc tgttgcactg gccctgcgat ttcgatctgt ttctgatgaa cattttccca
     3721 tactgtacct gcatttctta cgtgaatagc tgtctcaatc ctttcctgta cgccttcttc
     3781 gaccctagat ttcggcaggc ctgcacaagc atgctgtgtt gcggccaatc aaggtgtgct
     3841 ggcactagtc attccagctc aggcgaaaaa agcgcatctt acagctccgg acattcccag
     3901 ggcccaggtc ctaacatggg caaaggcggt gagcagatgc acgaaaaatc cattccttat
     3961 agtcaggaga ctctggtggt tgacatcgat accggtggac gcaccggtga attcgatgag
     4021 gccagcacca ccgtgtctaa gaccgagact tctcaggtgg cccctgccta actcgagtct
     4081 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     4141 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     4201 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     4261 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     4321 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     4381 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4441 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4501 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4561 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     4621 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4681 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     4741 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     4801 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     4861 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     4921 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     4981 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     5041 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     5101 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     5161 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     5221 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     5281 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     5341 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     5401 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     5461 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     5521 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     5581 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     5641 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     5701 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     5761 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     5821 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     5881 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     5941 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     6001 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     6061 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     6121 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     6181 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     6241 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     6301 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     6361 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     6421 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     6481 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     6541 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     6601 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     6661 aatacggtta tccacagaat
//
