LOCUS       sequence-430910-        6875 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 6875)
  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 6875)
  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: HRH3-DuET
FEATURES             Location/Qualifiers
     source          1..6875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     236..255
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(474..578)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(653..672)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        844..1223
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1224..1427
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1378..1398
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1472..1491
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1472..1490
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3075..3092)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3081..3305
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3351..3779
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3438..3457)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3648..3669
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3788..3808)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3793..4122
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3973..4108
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4035..4054
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4189..4983
                     /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(4243..4262)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4853..4872
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5157..5278
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5194..5213)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5248..5267
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5327..5343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5327..5343)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5340..5362)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5351..5367
                     /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(5375..5381,5382..5399,5400..5405))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5420..5441
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5558..5575)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5729..6317)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5809..5828)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6488..6875,1..404,405..473))
                     /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 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
       61 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
      121 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
      181 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
      241 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
      301 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
      361 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
      421 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
      481 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
      541 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
      601 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
      661 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
      721 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac atttgcatga
      781 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
      841 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      901 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      961 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     1021 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     1081 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     1141 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     1201 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     1261 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     1321 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     1381 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     1441 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     1501 ctggctagcg tttaaactta agcttagcgg ccgcgccacc atgaagacga tcatcgccct
     1561 gagctacatc ttctgcctgg tattcgccga ctacaaggac gatgatgacg ccagcatcga
     1621 tatggagaga gcaccacctg acggaccact gaatgcatct ggggcccttg caggagaagc
     1681 ggcagctgct ggcggagcca ggggattctc tgcagcttgg accgctgtgc tggccgccct
     1741 gatggctctc ctgatcgtcg ccacggtact ggggaacgct ctggtgatgc tggctttcgt
     1801 cgcagattca agtctgcgaa cccagaataa cttcttcctt ctcaacttgg ctattagtga
     1861 ttttctggtg ggcgcgttct gtatccctct gtacgtgccc tacgtgttga ccggcagatg
     1921 gacctttgga cgaggcctgt gcaagctgtg gttggttgtt gattacttgt tgtgtactag
     1981 cagcgcattt aacattgtcc tgatttctta cgaccgcttt ttgtccgtaa ccagggcggt
     2041 cagctatcgc gcgcagcagg gagacacacg cagggccgtt cggaaaatgc tgttggtttg
     2101 ggtgctggct ttcctccttt atggcccagc gatcttgtcc tgggagtatc tgagcggcgg
     2161 gtcctcaatt cccgagggcc attgctacgc tgagttcttc tataattggt actttctgat
     2221 tacagcttct acactggagt tcttcacacc ctttctgtct gtaactttct ttaatctgtc
     2281 tatatatctg aacattcaac ggagaactcg actccggctg gatggagcta gggaggccgc
     2341 cggtcctgag ccaccgcctg aggcacagcc atcaccccct cctccacctg gttgctgggg
     2401 atgttggcaa aagggccacg gggaggctat gccattgcat cgctatggcg ttggtgaagc
     2461 cgcagtcggt gcagaggcag gagaggctac cctgggaggg ggtggtgggg gcggttccgt
     2521 cgcctctcca actagctcca gtggaagtag cagtagaggt accgagcgcc cccgctctct
     2581 taaaaggggg tcaaagccca gtgccagctc agccagcctt gagaaaagaa tgaaaatggt
     2641 gtctcagtcc tttacccaaa ggttcaggct ctcaagagat cgcaaagtcg ctaaaagcct
     2701 ggcggtgatc gtcagcattt tcggactctg ttgggctcct tacaccctgc tgatgatcat
     2761 tagggcagcg tgccatggcc attgtgtccc cgattattgg tatgagactt ctttttggct
     2821 tttgtgggca aacagcgccg tcaatcccgt cctgtacccc ctgtgccatc actctttcag
     2881 aagggccttt acaaaactcc tgtgtcccca gaaactgaag atccaaccac acagtagcct
     2941 cgaacattgt tggaagatcg ataccggtgg acgcaccggt gaattcgatg aggccagcac
     3001 caccgtgtct aagaccgaga cttctcaggt ggcccctgcc taatctagag ggcccgttta
     3061 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3121 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3181 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3241 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3301 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     3361 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3421 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3481 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3541 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3601 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3661 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3721 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     3781 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3841 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3901 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     3961 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4021 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     4081 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     4141 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     4201 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4261 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4321 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     4381 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4441 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     4501 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4561 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     4621 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     4681 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     4741 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     4801 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     4861 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     4921 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     4981 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     5041 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     5101 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     5161 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5221 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5281 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5341 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5401 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5461 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5521 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5581 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5641 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5701 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5761 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5821 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5881 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5941 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6001 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6061 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6121 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acggtatttg
     6181 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6241 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6301 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6361 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6421 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6481 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6541 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6601 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6661 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6721 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6781 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6841 cttcattcag ctccggttcc caacgatcaa ggcga
//
