LOCUS       sequence-431815-        6632 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 6632)
  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 6632)
  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: HTR1F-DuET
FEATURES             Location/Qualifiers
     source          1..6632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     446..465
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(684..788)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(863..882)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1054..1433
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1434..1637
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1588..1608
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1682..1701
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1682..1700
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3042..3059)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3048..3272
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3318..3746
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3405..3424)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3615..3636
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3755..3775)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3760..4089
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3940..4075
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4002..4021
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4156..4950
                     /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(4210..4229)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4820..4839
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5124..5245
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5161..5180)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5215..5234
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5294..5310)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5294..5310)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5307..5329)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5318..5334
                     /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(5342..5348,5349..5366,5367..5372))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5387..5408
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5525..5542)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5696..6284)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5776..5795)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6455..6632,1..614,615..683))
                     /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"
ORIGIN
        1 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
       61 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
      121 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
      181 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
      241 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
      301 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
      361 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
      421 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
      481 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
      541 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
      601 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
      661 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
      721 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
      781 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
      841 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
      901 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
      961 caaggcaagg cttgaccgac atttgcatga agaatctgct tagggttagg cgttttgcgc
     1021 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
     1081 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     1141 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     1201 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     1261 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     1321 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     1381 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
     1441 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     1501 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
     1561 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
     1621 tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa
     1681 ttaatacgac tcactatagg gagacccaag ctggctagcg tttaaactta agcttagcgg
     1741 ccgcgccacc atgaagacga tcatcgccct gagctacatc ttctgcctgg tattcgccga
     1801 ctacaaggac gatgatgacg ccagcatcga tatggatttt ctgaattcct ctgaccagaa
     1861 cctcacatct gaggagctgc tgaacagaat gccgtccaag atcctggtca gccttacctt
     1921 gagcgggttg gctctgatga ctaccacgat caatagtctc gtgattgcgg caatcatcgt
     1981 gacaaggaaa ctgcaccacc ccgcaaacta tcttatctgc agtttggcag tgaccgactt
     2041 cctcgtggcc gtgctggtca tgcctttctc aatcgtctac attgtcagag agtcctggat
     2101 aatgggacaa gtcgtgtgcg acatctggct gagcgtggat atcacctgct gcacttgctc
     2161 tatcctgcat cttagtgcca tcgccctcga tcggtatcgg gccattaccg acgcagtgga
     2221 gtacgccagg aaacggaccc ctaaacatgc cggcattatg atcacaatcg tatggataat
     2281 ctccgttttt atatccatgc cacccctttt ttggcgccat cagggcacct cccgcgatga
     2341 tgaatgcatc attaaacacg accatatagt tagcacgata tatagtactt tcggagcttt
     2401 ttacatcccc ttggcactca ttctgatact ctattataaa atttacagag cggctaagac
     2461 cctgtatcac aagaggcaag catccagaat tgccaaagaa gaagtgaatg ggcaggtcct
     2521 gcttgagtct ggggagaaat ccactaagtc agttagtacc tcatatgtac tggaaaaaag
     2581 cttgagcgac ccatctactg actttgacaa aatccatagt accgtgagga gcttgcgaag
     2641 cgagtttaaa catgagaagt catggaggcg ccaaaagata tcagggacac gagaacgaaa
     2701 ggccgccaca actctgggac tcatcctcgg cgctttcgtc atctgctggc ttcccttctt
     2761 tgtaaaagag ctggttgtaa acgtctgcga taaatgcaag ataagcgagg agatgtctaa
     2821 tttcttggca tggctgggat acttgaattc acttatcaat ccccttatct ataccatctt
     2881 caacgaggat tttaaaaaag cgtttcagaa gcttgttcgc tgcaggtgta tcgataccgg
     2941 tgaattcgat gaggccagca ccaccgtgtc taagaccgag acttctcagg tggcccctgc
     3001 ctaactcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     3061 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3121 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3181 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     3241 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     3301 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3361 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3421 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3481 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3541 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3601 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3661 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3721 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     3781 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3841 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3901 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3961 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4021 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     4081 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     4141 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     4201 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     4261 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     4321 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     4381 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     4441 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     4501 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     4561 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     4621 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     4681 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     4741 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     4801 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4861 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4921 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4981 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     5041 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5101 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     5161 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5221 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     5281 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5341 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5401 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5461 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5521 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5581 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5641 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5701 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5761 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5821 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5881 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5941 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6001 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6061 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6121 acggctacac tagaagaacg gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6181 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6241 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6301 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6361 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6421 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6481 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6541 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6601 cacgctcacc ggctccagat ttatcagcaa ta
//
