LOCUS       sequence-430827-        6608 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 6608)
  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 6608)
  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: GPR32-DuET
FEATURES             Location/Qualifiers
     source          1..6608
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     468..487
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     721..738
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    855..876
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(891..896,897..914,915..921)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    929..945
                     /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)."
     primer_bind     934..956
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     953..969
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     953..969
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1018..1139
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1029..1048)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1083..1102
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1313..2107)
                     /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(1424..1443)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2034..2053
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2174..2503)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2188..2323
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2242..2261)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2488..2508
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2517..2945)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2627..2648)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2839..2858
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2991..3215
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3204..3221
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4538..4557)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4539..4557)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4602..4805)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4631..4651)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4806..5185
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5357..5376
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5451..5555
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5556..5624,5625..6416)
                     /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     complement(5774..5793)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6587..6608,1..567)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
       61 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
      121 accgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
      181 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
      241 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
      301 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
      361 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
      421 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
      481 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
      541 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
      601 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
      661 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
      721 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
      781 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      841 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
      901 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
      961 agctatgacc atgattacgc caagctctag ctagaggtcg acggtataca gacatgataa
     1021 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     1081 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg
     1141 gggtgggcga agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc
     1201 cggaaaacga ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt
     1261 gatggcaggt tgggcgtcgc ttggtcggtc atttcgaacc ccagagtccc gctcagaaga
     1321 actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa
     1381 gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca
     1441 acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa
     1501 agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat
     1561 cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct
     1621 gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc
     1681 gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca
     1741 gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca
     1801 ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa
     1861 cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct
     1921 cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc
     1981 cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt
     2041 catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt
     2101 caatcatgcg aaacgatcct catcctgtct cttgatcaga tccgaaaatg gatatacaag
     2161 ctcccgggag ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga
     2221 atagctcaga ggcagaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat
     2281 ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg
     2341 ggactatggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     2401 ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc
     2461 ctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac agaattaatt
     2521 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     2581 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     2641 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     2701 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     2761 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     2821 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     2881 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     2941 cgcgtgggga taccccctag agccccagct ggttctttcc gcctcagaag ccatagagcc
     3001 caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc
     3061 caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt
     3121 ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg
     3181 gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc gggtttaaac
     3241 gggccctcta gactcgagtt aggcaggggc cacctgagaa gtctcggtct tagacacggt
     3301 ggtgctggcc tcatcgaatt caccggtgcg tccaccggta tcgatctccc gtggggcatt
     3361 gccgcgtgga caagaactca aaaactcttc ctcgccgaag gcccgcgcga gggctgatgt
     3421 cagactctga aagaattttt cctggaagtc cctcccgacg aaaacgtaca ggaagggatt
     3481 cagagaagag ttgacgcatc cgagggcgaa actggcctga aggatgagga gcatgcgtgg
     3541 gtgataaatc tccttcaaca tcaccctgcg ccacaagtgg accaggagaa ccacgttgaa
     3601 tggggaccag aaaataaaga aagcagaaac caggaccagc agcagcctct tgggcctgtt
     3661 ggcatgcacc cagccctctc tcagcagctt ggctctaatg aggtgtgcac aagtgccaat
     3721 gatggccagt gggcccaaaa atcccagcaa aaaatgacca atagtgccga tgatgtggcc
     3781 ctccacgaca ccctcaatcc agatttgtgc tgtctcgttg tcggagttaa aggccagata
     3841 gcagtgggta caaccgttcc acttcctcgt agttctgaac ttcagatggg cgctgcacag
     3901 cgctgcggcc aggagccaca cgccgaaagc caaccagctg gctctttgta cggtgcggtg
     3961 gttcaaagcc cacacagggt aaagcacgga gatacagcgg tccacactga tgaacacaag
     4021 caggcagttt gaagcgaagt agctgagaaa cacgaaagta atgtagagct tgcaagccca
     4081 ttcacccagc agccactggc ggctcacgat gtagtacatg gcgattggga gactcagaga
     4141 cagcataaaa tcagccaggg cgaggtggaa aaagcacact gttgatacgg tccgcgccat
     4201 cctgaacaca gtcatccaca gcaccaggcc attgccgagt acacccacca caatgctagc
     4261 agacagtatc accactgtca agggcctcag actgccgacc tcttcgctca ggcagcctga
     4321 gctgttcatc tttcgtgaac aggatctgtc tctagtaagg accccaggtt gccggtcgct
     4381 acaaccgcga gtaccttcac ttacaccgtt catatcgatg ctggcgtcat catcgtcctt
     4441 gtagtcggcg aataccaggc agaagatgta gctcagggcg atgatcgtct tcatggtggc
     4501 gctaagctta agtttaaacg ctagccagct tgggtctccc tatagtgagt cgtattaatt
     4561 tcgataagcc agtaagcagt gggttctcta gttagccaga gagctctgct tatatagacc
     4621 tcccaccgta cacgcctacc gcccatttgc gtcaatgggg cggagttgtt acgacatttt
     4681 ggaaagtccc gttgattttg gtgccaaaac aaactcccat tgacgtcaat ggggtggaga
     4741 cttggaaatc cccgtgagtc aaaccgctat ccacgcccat tgatgtactg ccaaaaccgc
     4801 atcaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg aaagtcccat
     4861 aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg
     4921 ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac
     4981 tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac atacgtcatt
     5041 attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt
     5101 tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat
     5161 taataactag tcaataatca atgtcaacgc gtatatctgg cccgtacatc gcgaagcagc
     5221 gcaaaacgcc taaccctaag cagattcttc atgcaaatgt cggtcaagcc ttgccttgtt
     5281 gtagcttaaa ttttgctcgc gcactactca gcgacctcca acacacaagc agggagcaga
     5341 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca taggggatcg
     5401 ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5461 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5521 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5581 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5641 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5701 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5761 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5821 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5881 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5941 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6001 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6061 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6121 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6181 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6241 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6301 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6361 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6421 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6481 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6541 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6601 ttctgcgc
//
