LOCUS       sequence-430850-        7001 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 7001)
  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 7001)
  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: HRH1-DuET
FEATURES             Location/Qualifiers
     source          1..7001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     255..274
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(493..597)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(672..691)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        863..1242
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1243..1446
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1397..1417
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1491..1510
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1491..1509
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3220..3237)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3226..3450
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3496..3924
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3583..3602)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3793..3814
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3933..3953)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3938..4267
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4118..4253
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4180..4199
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4334..5128
                     /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(4388..4407)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4998..5017
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5302..5423
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5339..5358)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5393..5412
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5472..5488)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5472..5488)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5485..5507)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5496..5512
                     /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(5520..5526,5527..5544,5545..5550))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5565..5586
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5703..5720)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5874..6462)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6633..7001,1..423,424..492))
                     /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 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
       61 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
      121 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
      181 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
      241 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
      301 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
      361 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
      421 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
      481 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
      541 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
      601 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
      661 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
      721 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
      781 ttgaccgaca tttgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
      841 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
      901 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      961 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     1021 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     1081 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     1141 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     1201 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     1261 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     1321 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     1381 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     1441 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact
     1501 cactataggg agacccaagc tggctagcgt ttaaacttaa gcttagcgcc accatgaaga
     1561 cgatcatcgc cctgagctac atcttctgcc tggtattcgc cgactacaag gacgatgatg
     1621 acgccagcat cgatatgtca ctgcctaact catcttgcct cctggaggac aagatgtgcg
     1681 aggggaacaa gaccactatg gcatcacccc agttgatgcc cctcgtggtc gtattgtcta
     1741 caatctgcct ggttaccgtg ggattgaatt tgcttgtgtt gtatgctgtg cggagcgaaa
     1801 ggaaacttca tactgtgggc aacctgtaca ttgtgagttt gtccgttgca gatctgatcg
     1861 tgggcgcggt cgttatgcct atgaatattc tgtacctgct gatgagcaaa tggtctctcg
     1921 gaaggccact gtgtctgttt tggttgtcca tggactacgt ggcaagcact gctagcatct
     1981 ttagtgtgtt catcctgtgt atcgacagat acagatctgt ccagcagcct ttgcgatatc
     2041 tgaagtatag gaccaagacc cgcgcgtcag caaccatcct cggcgcctgg tttctttctt
     2101 tcctgtgggt gatccccata ttggggtgga accacttcat gcagcaaacg tcagttcgca
     2161 gggaggataa gtgcgaaaca gacttctacg acgtcacatg gtttaaggtg atgacagcca
     2221 tcatcaactt ttatctgccc actttgttga tgctgtggtt ctacgctaag atatacaagg
     2281 ccgtgagaca gcactgccaa catcgggaac tgataaatcg gtctttgcca tcatttagtg
     2341 aaatcaagct ccgcccagag aatcctaaag gtgatgcaaa gaagcccgga aaggaaagtc
     2401 cttgggaagt cctgaaaaga aagcccaagg acgcgggcgg cgggtcagta cttaagagtc
     2461 cttctcagac tccaaaggag atgaaatcac ccgtggtgtt cagccaggag gacgatcggg
     2521 aggtggacaa actctactgc ttccccctgg acattgtgca catgcaggct gcagctgagg
     2581 gcagctcaag ggattacgtc gcggtgaatc gcagtcacgg tcaactgaaa actgacgagc
     2641 aaggactgaa tactcatggc gcctcagaga tttccgaaga ccagatgctg ggggactctc
     2701 agtctttttc caggacggac agcgatacca ctaccgagac tgcacctggg aaaggcaagc
     2761 tccggtcagg aagcaatacc ggcctggact atatcaagtt cacctggaaa agactgagaa
     2821 gccactcccg gcaatatgtt tcaggcctgc acatgaaccg ggaacgcaaa gccgcaaaac
     2881 aactcgggtt cattatggcc gctttcatct tgtgctggat accctacttc atattcttca
     2941 tggtgattgc tttctgcaag aactgttgta atgagcacct gcacatgttt acaatttggc
     3001 tgggctatat aaactccacc cttaatccgc tgatttaccc cctttgtaac gaaaacttta
     3061 aaaagacatt caagcggatc ttgcatataa ggtccatcga taccggtgga cgcaccggtg
     3121 aattcgatga ggccagcacc accgtgtcta agaccgagac ttctcaggtg gcccctgcct
     3181 aactcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     3241 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3301 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3361 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     3421 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     3481 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3541 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3601 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3661 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3721 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3781 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3841 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3901 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     3961 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4021 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4081 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4141 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4201 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     4261 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     4321 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     4381 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     4441 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4501 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4561 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4621 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4681 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4741 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4801 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4861 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4921 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4981 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     5041 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     5101 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     5161 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     5221 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5281 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     5341 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5401 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     5461 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5521 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5581 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5641 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5701 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5761 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5821 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5881 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5941 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6001 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6061 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6121 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6181 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6241 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6301 ggctacacta gaagaacggt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6361 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6421 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6481 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6541 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6601 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6661 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6721 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6781 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6841 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6901 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6961 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg g
//
