LOCUS       sequence-430854-        6710 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 6710)
  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 6710)
  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: HTR1B-DuET
FEATURES             Location/Qualifiers
     source          1..6710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(37..56)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(716..1507,1508..1576))
                     /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     1339..1358
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1577..1681)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1756..1775)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1947..2326
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2327..2530
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2481..2501
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2575..2594
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2575..2593
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4013..4030)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4019..4243
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4289..4717
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4376..4395)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4586..4607
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4726..4746)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4731..5060
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4911..5046
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4973..4992
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5127..5921
                     /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(5181..5200)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5791..5810
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6095..6216
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6132..6151)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6186..6205
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6265..6281)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6265..6281)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6278..6300)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6289..6305
                     /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(6313..6319,6320..6337,6338..6343))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6358..6379
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6496..6513)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6667..6710,1..545))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
       61 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
      121 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
      181 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
      241 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
      301 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
      361 tcttgaagtg gtggcctaac tacggctaca ctagaagaac ggtatttggt atctgcgctc
      421 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
      481 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
      541 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
      601 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
      661 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
      721 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
      781 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
      841 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
      901 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
      961 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     1021 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     1081 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     1141 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     1201 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     1261 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     1321 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     1381 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     1441 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     1501 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     1561 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     1621 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     1681 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     1741 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     1801 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     1861 aggcttgacc gacatttgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     1921 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
     1981 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     2041 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     2101 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     2161 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     2221 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     2281 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     2341 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     2401 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     2461 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     2521 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
     2581 gactcactat agggagaccc aagctggcta gcgtttaaac ttaagcttag cgccaccatg
     2641 aagacgatca tcgccctgag ctacatcttc tgcctggtat tcgccgacta caaggacgat
     2701 gatgacgcca gcatcgatat ggaggagcct ggcgctcaat gcgctccacc tccccctgct
     2761 gggtctgaga cttgggtgcc acaggctaat ctgtcaagtg caccaagcca gaactgctcc
     2821 gcaaaggatt acatatatca agacagtata agtctgcctt ggaaagtgct tctcgtgatg
     2881 cttctcgccc tgatcacctt ggccacaaca ctctctaacg ccttcgttat agcaaccgtg
     2941 tatcgaactc ggaagttgca tacccccgcc aattatttga ttgcctccct ggccgtgacc
     3001 gatcttctgg tttcaatcct ggtcatgccg atctcaacta tgtacaccgt gactggccga
     3061 tggacacttg ggcaggtggt gtgcgacttc tggttgagct cagacatcac ctgctgtacc
     3121 gcctctattc tccacctgtg cgtcattgct ctggataggt actgggccat caccgacgcc
     3181 gttgagtata gcgctaagcg gacccctaaa agggccgccg tgatgattgc cttggtgtgg
     3241 gtcttcagca tttcaatctc tctcccaccg tttttctgga gacaggctaa agcagaagag
     3301 gaggtctccg aatgtgtggt caacaccgac catatactgt acactgtata ctcaaccgtc
     3361 ggcgccttct actttcctac cctcctcctg atagcactct acggccggat ctatgttgag
     3421 gcaagaagcc ggatcttgaa gcagacgccc aaccgaacag gcaaacgcct cacaagggct
     3481 caactcatca cagattcacc cggctcaaca agctccgtta cctcaatcaa ttctcgggtc
     3541 cccgatgtcc cgagcgagtc cggttctcct gtatacgtga accaggttaa ggtgcgcgta
     3601 tctgacgcct tgcttgaaaa gaaaaaattg atggccgcac gcgaaaggaa agccacaaag
     3661 actctcggta ttatcctcgg agcctttatt gtgtgttggc tgcctttttt cattatcagc
     3721 ctggtgatgc ctatctgcaa agatgcgtgc tggttccacc tcgctatctt cgattttttt
     3781 acttggctgg ggtacctgaa ctctttgatc aatcccatta tttatacaat gtctaacgaa
     3841 gactttaagc aggcattcca taagttgatt cggttcaagt gtacatcaat cgataccggt
     3901 ggacgcaccg gtgaattcga tgaggccagc accaccgtgt ctaagaccga gacttctcag
     3961 gtggcccctg cctaactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact
     4021 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     4081 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     4141 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     4201 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     4261 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     4321 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4381 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4441 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4501 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4561 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4621 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4681 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     4741 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4801 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4861 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4921 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4981 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     5041 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     5101 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     5161 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     5221 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     5281 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     5341 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     5401 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     5461 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     5521 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     5581 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     5641 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     5701 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     5761 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     5821 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     5881 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     5941 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     6001 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     6061 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     6121 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     6181 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     6241 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     6301 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     6361 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6421 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6481 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     6541 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6601 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6661 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
//
