LOCUS       sequence-430825-        6665 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 6665)
  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 6665)
  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: GPR27-DuET
FEATURES             Location/Qualifiers
     source          1..6665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(570..1361,1362..1430))
                     /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     1193..1212
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1431..1535)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1610..1629)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1801..2180
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2181..2384
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2335..2355
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2429..2448
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2429..2447
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3822..3839)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3828..4052
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4098..4526
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4185..4204)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4395..4416
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4535..4555)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4540..4869
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4720..4855
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4782..4801
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4936..5730
                     /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(4990..5009)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5600..5619
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5904..6025
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5941..5960)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5995..6014
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6074..6090)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6074..6090)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6087..6109)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6098..6114
                     /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(6122..6128,6129..6146,6147..6152))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6167..6188
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6305..6322)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6476..6665,1..399))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6556..6575)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
       61 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
      121 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
      181 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
      241 tacactagaa gaacggtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
      301 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
      361 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
      421 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
      481 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
      541 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
      601 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
      661 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
      721 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
      781 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
      841 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
      901 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
      961 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     1021 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     1081 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     1141 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     1201 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     1261 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     1321 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     1381 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     1441 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     1501 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     1561 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     1621 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     1681 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacatt tgcatgaaga
     1741 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
     1801 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     1861 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     1921 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     1981 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     2041 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     2101 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     2161 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     2221 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     2281 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     2341 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
     2401 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
     2461 gctagcgttt aaacttaagc ttagcgccac catgaagacg atcatcgccc tgagctacat
     2521 cttctgcctg gtattcgccg actacaagga cgatgatgac gccagcatcg atatggccaa
     2581 tgccagtgag ccaggcggct ccgggggtgg cgaggccgca gccctggggt tgaaactggc
     2641 cacactgtcc ctgcttcttt gcgtgagcct cgctggtaac gtgctgttcg cactgctcat
     2701 cgtgcgcgag aggtcactgc accgcgcacc ctattacctg cttctcgatc tgtgtctggc
     2761 cgacggtctc agggctttgg catgcttgcc ggccgtcatg ctcgccgcga gaagggctgc
     2821 tgcagcagcc ggagcaccac ccggagcact cggctgcaaa ctgttggctt tcctcgccgc
     2881 cctcttctgt tttcatgctg cttttctcct cctgggcgtg ggcgtcacta gatatctggc
     2941 tatcgcccac caccgattct acgccgaacg gctggcgggt tggccatgtg ctgcaatgct
     3001 tgtctgtgcc gcctgggcac ttgctctggc tgcagcattt ccccctgtgc tggacggagg
     3061 gggggacgat gaggatgcgc catgcgcact ggaacagcgg cccgacggag ctcctggcgc
     3121 cctcgggttt ctgctcttgc tggctgtagt ggtcggggcc acacacttgg tatatttgcg
     3181 gctgctgttc ttcatccacg acagacgcaa gatgcggcct gccaggctgg tgccagcggt
     3241 gtcccacgat tggacgttcc acgggcctgg cgcaacgggt caggctgcag ccaactggac
     3301 agccggattt ggaagaggac ctaccccccc cgctctggtg ggaattaggc ccgccggacc
     3361 aggaagggga gcccgcaggc ttctggttct cgaagaattt aagactgaaa agcgactgtg
     3421 caaaatgttt tacgcggtga cgctgttgtt cctcctgctg tggggtccat atgtggtggc
     3481 ctcttatctc agagtgctgg tgaggccagg agctgtacct caggcctacc tcaccgccag
     3541 cgtatggctg actttcgcgc aggccggaat taatcctgtg gtgtgctttc tttttaacag
     3601 ggaactccgg gattgcttcc gggcacaatt cccatgctgc cagtccccga ggaccacaca
     3661 ggcaactcac ccatgcgact tgaaaggcat cggtctgatc gataccggtg gacgcaccgg
     3721 tgaattcgat gaggccagca ccaccgtgtc taagaccgag acttctcagg tggcccctgc
     3781 ctaactcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     3841 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3901 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3961 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     4021 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     4081 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4141 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     4201 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4261 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4321 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4381 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4441 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4501 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     4561 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4621 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4681 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     4741 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4801 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     4861 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     4921 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     4981 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     5041 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     5101 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     5161 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     5221 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     5281 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     5341 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     5401 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     5461 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     5521 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     5581 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     5641 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     5701 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     5761 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     5821 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5881 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     5941 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     6001 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     6061 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     6121 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     6181 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6241 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     6301 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6361 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     6421 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6481 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6541 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6601 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6661 cgctt
//
