LOCUS       sequence-430860-        6611 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 6611)
  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 6611)
  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: HTR5-DuET
FEATURES             Location/Qualifiers
     source          1..6611
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(723..740)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    729..953
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      999..1427
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1086..1105)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1296..1317
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1436..1456)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1441..1770
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1621..1756
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1683..1702
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1837..2631
                     /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(1891..1910)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2501..2520
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2805..2926
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2842..2861)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2896..2915
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2975..2991)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2975..2991)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2988..3010)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2999..3015
                     /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(3023..3029,3030..3047,3048..3053))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3068..3089
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3206..3223)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3377..3965)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3457..3476)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4136..4927,4928..4996))
                     /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     4759..4778
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4997..5101)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5176..5195)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5367..5746
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5747..5950
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5901..5921
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5995..6014
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5995..6013
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 caatgttatg atcgctctga cctgggccct gagcgctgtg atcagccttg cgccgctgct
       61 tttcggatgg ggagagacat attcagaggg atcagaggaa tgccaggtca gtcgggagcc
      121 ttcctatgcg gtgtttagca ctgtcggcgc tttttatctc ccactgtgtg tcgtgctgtt
      181 cgtctattgg aaaatttata aggctgccaa gtttcgggta ggaagccgga aaacgaactc
      241 cgtgagcccg atcagtgaag ctgtggaagt gaaggactct gctaagcagc cacagatggt
      301 gttcacggtc aggcatgcca ctgtgacatt ccagcccgaa ggggacacct ggagagaaca
      361 gaaagaacag agggcagccc tgatggtcgg gatcctgatc ggtgtgtttg tcctctgctg
      421 gatacccttc ttcctgacag aacttatcag ccccctctgt agctgtgaca ttcccgctat
      481 ttggaaaagc atctttctgt ggcttggata cagtaactct tttttcaatc ctctgatata
      541 cacagcattc aataaaaact acaactccgc attcaaaaat ttcttttcac ggcagcatat
      601 cgataccggt ggacgcaccg gtgaattcga tgaggccagc accaccgtgt ctaagaccga
      661 gacttctcag gtggcccctg cctaactcga gtctagaggg cccgtttaaa cccgctgatc
      721 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
      781 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
      841 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
      901 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
      961 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     1021 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     1081 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1141 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1201 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1261 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     1321 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     1381 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     1441 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     1501 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     1561 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     1621 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     1681 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     1741 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     1801 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     1861 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     1921 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     1981 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     2041 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     2101 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     2161 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     2221 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     2281 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     2341 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     2401 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     2461 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     2521 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     2581 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     2641 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     2701 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     2761 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     2821 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2881 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     2941 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     3001 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     3061 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     3121 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3181 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3241 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     3301 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3361 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3421 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3481 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3541 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3601 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3661 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3721 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3781 tcttgaagtg gtggcctaac tacggctaca ctagaagaac ggtatttggt atctgcgctc
     3841 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3901 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3961 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4021 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4081 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     4141 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4201 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     4261 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     4321 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     4381 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     4441 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4501 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4561 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4621 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4681 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4741 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     4801 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     4861 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     4921 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     4981 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5041 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5101 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     5161 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     5221 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     5281 aggcttgacc gacatttgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     5341 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
     5401 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     5461 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     5521 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     5581 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     5641 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     5701 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     5761 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     5821 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     5881 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     5941 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
     6001 gactcactat agggagaccc aagctggcta gcgtttaaac ttaagcttag cgccaccatg
     6061 aagacgatca tcgccctgag ctacatcttc tgcctggtat tcgccgacta caaggacgat
     6121 gatgacgcca gcatcgatat ggacctgccc gtcaacttga cgagtttcag tctgtccact
     6181 ccaagtcctc ttgagactaa tcactctctt ggaaaggacg acttgaggcc ttccagccct
     6241 ctcctctccg tgtttggggt gctgattctt accctgctcg gatttctggt agccgctact
     6301 ttcgcatgga acctgttggt gctggcaacc attctccgcg tgcggacatt ccacagggtt
     6361 ccccataatc ttgtggcatc catggcagtg agtgacgtac tggtagcagc actggtgatg
     6421 cctctttctc tcgtccatga actcagcggg cggagatggc agcttggacg aagactttgt
     6481 cagttgtgga ttgcatgtga cgtactttgc tgtactgctt ccatttggaa tgtcaccgcc
     6541 attgcccttg accgctactg gtccatcact cgccatatgg agtatacctt gaggacccga
     6601 aaatgtgtct c
//
