LOCUS       sequence-430848-        6815 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 6815)
  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 6815)
  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: HCTR1-DuET
FEATURES             Location/Qualifiers
     source          1..6815
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      186..774
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     675..694
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     928..945
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1062..1083
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1098..1103,1104..1121,1122..1128)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1136..1152
                     /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     1141..1163
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1160..1176
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1160..1176
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1225..1346
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1236..1255)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1290..1309
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1520..2314)
                     /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(1631..1650)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2241..2260
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2381..2710)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2395..2530
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2695..2715
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2724..3152)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2834..2855)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3046..3065
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3198..3422
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3411..3428
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4952..4971)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4953..4971)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5016..5219)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5045..5065)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5220..5599
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5771..5790
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5865..5969
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5970..6038,6039..6815,1..15)
                     /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(6188..6207)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
       61 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
      121 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
      181 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
      241 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
      301 ggcttcagca gagcgcagat accaaatacc gttcttctag tgtagccgta gttaggccac
      361 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
      421 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
      481 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
      541 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
      601 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
      661 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
      721 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
      781 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
      841 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
      901 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
      961 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     1021 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     1081 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     1141 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctctagcta
     1201 gaggtcgacg gtatacagac atgataagat acattgatga gtttggacaa accacaacta
     1261 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     1321 ccattataag ctgcaataaa caagttgggg tgggcgaaga actccagcat gagatccccg
     1381 cgctggagga tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag
     1441 aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt
     1501 tcgaacccca gagtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct
     1561 gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa
     1621 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca
     1681 gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc
     1741 aggcatcgcc atgggtcacg acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg
     1801 cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa
     1861 gaccggcttc catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg
     1921 ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt
     1981 tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca
     2041 gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg
     2101 tggccagcca cgatagccgc gctgcctcgt cctgcagttc attcagggca ccggacaggt
     2161 cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag
     2221 agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg
     2281 gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt
     2341 gatcagatcc gaaaatggat atacaagctc ccgggagctt tttgcaaaag cctaggcctc
     2401 caaaaaagcc tcctcactac ttctggaata gctcagaggc agaggcggcc tcggcctctg
     2461 cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta
     2521 ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc
     2581 tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat
     2641 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta
     2701 actgacacac attccacaga attaattcgc gttaaatttt tgttaaatca gctcattttt
     2761 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     2821 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     2881 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     2941 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     3001 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     3061 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     3121 cgccgcgctt aatgcgccgc tacagggcgc gtggggatac cccctagagc cccagctggt
     3181 tctttccgcc tcagaagcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc
     3241 ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact
     3301 cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc
     3361 cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac
     3421 agtcgaggct gatcagcggg tttaaacggg ccctctagac tcgagttagg caggggccac
     3481 ctgagaagtc tcggtcttag acacggtggt gctggcctca tcgaattcac cggtgcgtcc
     3541 accggtatcg atcggaagta cggtggtgac tgacgtaagc accacatgtt cggatatctt
     3601 tgatatggaa caccggctct gcagggagag gctcttgtgg ctggcagagc ttctcgggct
     3661 tggagcttta agagagccgc aggggccgag gccaggcaga cagcaggaga aagcagcttt
     3721 aaattgttcc ctgaactttc ctgacaggaa attatagatg atcgggttgg cagcactgtt
     3781 ggcgtatacc agccagtggc taaaggtaaa acaggcataa acggcttcgc ggtcagaagc
     3841 ttgcctgaac attccgaata ctcgtttcag cacgttcagc acggagatcg gcaggtagca
     3901 caacgcaaag acaagcaaca cgaccatcag catttttgct gtctttctcc tggcccgcat
     3961 ctgcttgact tcggcgagga aggctcgtgc ccgaggctgt ggctcgccac tcagtccctg
     4021 ttccaggtca ccgagttggt cggacggtct cttccaattg cgcaccaggg cgctggttgt
     4081 accgggaatc tgtcgtcccc acaatttccg aaagatctga aagtatgcca tggccatcag
     4141 accgaggggg gcgaggtagg tgacaataaa gaagcagcta tggtagattt tagggtagag
     4201 atcatcggcc catctctcat cacacacgct gaaaaggcga gttctatttg caagctcggg
     4261 cagtacggaa gagcactcca taacggcggc ttgaggcacc attatagcca gagacacggc
     4321 ccagatgccc aggatgctac cccgggcgcg tctggcagtg ctcttgaaca gcagtgggtg
     4381 gcagattgca taccatctat caagggcgat gaagctgaga gtcagtactg ccacagacac
     4441 ggagacggcc tgcagatagg gaatcacctt gcagagggcg tgaccgaaga gccagctttc
     4501 agtaatgtca acgagcaaac tagcaggaag acagatggca gtgaccagca catctgcgag
     4561 tgacagattg acgataaagt aatttgtcac cgtccgcata tggtggtttc tccacactgc
     4621 gaggcataca agggtattgc ccacgagggc cacgacaaac actgccacgt aagcggctat
     4681 cagcacccat tcatactgct ttggataaag ataatcgcgc cacagatacc gaaggaactc
     4741 gtcttcgtag tcgggtggta ctggagaagg ttcccggcta cctggtggaa ctcccatctg
     4801 agcgcctggt gtggcactcg gctccatatc gatgctggcg tcatcatcgt ccttgtagtc
     4861 ggcgaatacc aggcagaaga tgtagctcag ggcgatgatc gtcttcatgg tggcgctaag
     4921 cttaagttta aacgctagcc agcttgggtc tccctatagt gagtcgtatt aatttcgata
     4981 agccagtaag cagtgggttc tctagttagc cagagagctc tgcttatata gacctcccac
     5041 cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag
     5101 tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg gagacttgga
     5161 aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa ccgcatcacc
     5221 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
     5281 atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
     5341 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
     5401 cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac
     5461 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
     5521 acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta ctattaataa
     5581 ctagtcaata atcaatgtca acgcgtatat ctggcccgta catcgcgaag cagcgcaaaa
     5641 cgcctaaccc taagcagatt cttcatgcaa atgtcggtca agccttgcct tgttgtagct
     5701 taaattttgc tcgcgcacta ctcagcgacc tccaacacac aagcagggag cagatactgg
     5761 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatagggg atcgggagat
     5821 ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5881 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5941 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     6001 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     6061 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     6121 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     6181 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     6241 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6301 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6361 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6421 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6481 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6541 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6601 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6661 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6721 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6781 gaaatagaca gatcgctgag ataggtgcct cactg
//
