LOCUS       sequence-430912-        6806 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 6806)
  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 6806)
  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: MCHR1-DuET
FEATURES             Location/Qualifiers
     source          1..6806
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     359..378
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(597..701)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(776..795)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        967..1346
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1347..1550
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1501..1521
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1595..1614
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1595..1613
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3129..3146)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3135..3359
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3405..3833
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3492..3511)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3702..3723
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3842..3862)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3847..4176
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4027..4162
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4089..4108
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4243..5037
                     /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(4297..4316)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4907..4926
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5211..5332
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5248..5267)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5302..5321
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5381..5397)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5381..5397)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5394..5416)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5405..5421
                     /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(5429..5435,5436..5453,5454..5459))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5474..5495
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5612..5629)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5783..6371)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5863..5882)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6542..6806,1..527,528..596))
                     /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"
ORIGIN
        1 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
       61 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
      121 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
      181 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
      241 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
      301 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
      361 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
      421 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
      481 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
      541 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
      601 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
      661 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
      721 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
      781 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      841 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacatttgca
      901 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      961 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     1021 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     1081 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     1141 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     1201 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     1261 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     1321 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     1381 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     1441 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     1501 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
     1561 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
     1621 aagctggcta gcgtttaaac ttaagcttag cggccgcgcc accatgaaga cgatcatcgc
     1681 cctgagctac atcttctgcc tggtattcgc cgactacaag gacgatgatg acgccagcat
     1741 cgatatgtca gtgggcgcca tgaaaaaggg cgtggggcgc gcggtgggac tggggggagg
     1801 ttcagggtgt caggcaactg aggaggatcc actgcctaat tgtggtgctt gtgcacctgg
     1861 gcaggggggg aggaggtgga gactcccgca gcccgcctgg gttgagggat cttccgcccg
     1921 gctgtgggag caagcaactg gaacaggctg gatggatctg gaagcctccc tgctgccgac
     1981 tggccctaac gcttccaata cctctgatgg ccctgataac ctcaccagtg ccgggtctcc
     2041 cccccgaacc ggctccatct cctacattaa cattattatg cctagcgtgt ttggcacgat
     2101 ttgtctcctt ggtattatcg ggaactctac agtgattttc gcagtggtga aaaaatcaaa
     2161 actgcattgg tgcaacaacg tgcccgatat atttatcatc aacctgagcg tggtcgatct
     2221 cctgttcctg cttggaatgc catttatgat ccatcagctc atgggcaacg gggtgtggca
     2281 ctttggcgaa acgatgtgca ccctcatcac tgcgatggat gccaactctc agttcacaag
     2341 tacctatatc cttacagcaa tggctataga ccgctatttg gccactgtac atccaatctc
     2401 aagcaccaaa ttccgcaagc ctagtgttgc aaccctggtc atctgccttc tctgggccct
     2461 cagcttcatt agcataactc cagtatggtt gtacgctagg ttgattccat ttcccggggg
     2521 cgcagtaggg tgcggtatca gattgccaaa tcctgatacc gatctgtact ggtttaccct
     2581 ttatcagttc tttcttgcct tcgccctgcc attcgtagtg atcaccgctg cttacgtgcg
     2641 gatcttgcag agaatgacct cttccgtggc accggcctca cagaggtcca tccggctcag
     2701 aactaaacgc gttaccagga ccgccatcgc catctgtctg gttttcttcg tctgctgggc
     2761 gccctactac gtgctccagc ttactcagct gtcaattagt cgccccaccc tcacttttgt
     2821 gtacctctat aatgccgcga ttagcctggg gtacgcgaat agttgcctca accctttcgt
     2881 ctacatcgtg ttgtgcgaga ctttcaggaa gagactggta ctgtccgtca agccagctgc
     2941 ccagggccag ctgcgggcgg tgagcaacgc acagaccgca gacgaggagc ggaccgagtc
     3001 caagggcacg atcgataccg gtggacgcac cggtgaattc gatgaggcca gcaccaccgt
     3061 gtctaagacc gagacttctc aggtggcccc tgcctaatct agagggcccg tttaaacccg
     3121 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3181 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3241 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3301 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3361 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     3421 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3481 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3541 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3601 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3661 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3721 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3781 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     3841 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3901 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3961 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     4021 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4081 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     4141 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     4201 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     4261 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     4321 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     4381 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     4441 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     4501 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     4561 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     4621 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     4681 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4741 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     4801 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4861 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4921 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4981 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     5041 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     5101 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     5161 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     5221 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     5281 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     5341 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     5401 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     5461 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     5521 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5581 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5641 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5701 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5761 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5821 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5881 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5941 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     6001 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6061 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6121 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6181 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacggta tttggtatct
     6241 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6301 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6361 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6421 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6481 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6541 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6601 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6661 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6721 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6781 agtctattaa ttgttgccgg gaagct
//
