LOCUS       sequence-430772-        6977 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 6977)
  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 6977)
  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: CHRM4-DuET
FEATURES             Location/Qualifiers
     source          1..6977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(651..1442,1443..1511))
                     /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     1274..1293
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1512..1616)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1691..1710)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1882..2261
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2262..2465
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2416..2436
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2510..2529
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2510..2528
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4215..4232)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4221..4445
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4491..4919
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4578..4597)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4788..4809
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4928..4948)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4933..5262
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5113..5248
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5329..6123
                     /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(5383..5402)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5993..6012
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6297..6418
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6334..6353)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6388..6407
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6467..6483)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6467..6483)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6480..6502)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6491..6507
                     /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(6515..6521,6522..6539,6540..6545))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6560..6581
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6698..6715)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6869..6977,1..480))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6949..6968)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
       61 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
      121 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
      181 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
      241 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
      301 aagtggtggc ctaactacgg ctacactaga agaacggtat ttggtatctg cgctctgctg
      361 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
      421 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
      481 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
      541 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      601 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      661 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      721 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      781 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      841 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      901 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      961 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     1021 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     1081 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     1141 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     1201 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     1261 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     1321 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     1381 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     1441 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1501 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1561 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1621 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     1681 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     1741 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     1801 tgaccgacat ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     1861 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     1921 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1981 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     2041 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     2101 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     2161 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     2221 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     2281 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     2341 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     2401 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     2461 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     2521 actataggga gacccaagct ggctagcgtt taaacttaag cttagcgcca ccatgaagac
     2581 gatcatcgcc ctgagctaca tcttctgcct ggtattcgcc gactacaagg acgatgatga
     2641 cgccagcatc gatatggcga atttcactcc tgttaatggt tccagtggca accagtctgt
     2701 tagactggtg acttctagct ctcacaacag gtatgagaca gttgaaatgg tcttcattgc
     2761 cactgtgaca ggctcactga gcctggtaac tgtcgttgga aacattctcg tgatgctgtc
     2821 catcaaagtg aacagacagc tgcagactgt caacaactat ttcctgtttt ccctggcctg
     2881 tgctgacctg ataataggcg ctttctcaat gaatctgtat accgtgtata tcatcaaagg
     2941 ttattggcct ctgggcgccg tcgtgtgtga tttgtggttg gcactcgact atgtggtatc
     3001 aaacgcttca gtgatgaatc tgcttataat ctcattcgat cgctatttct gtgtgactaa
     3061 acctctgact tacccagccc ggaggaccac aaaaatggcc ggcctcatga tcgccgccgc
     3121 atgggtcctg tcattcgtct tgtgggcacc ggctatcctc ttctggcagt ttgtggtggg
     3181 caagcggacc gtccctgaca atcagtgctt catccagttt ttgtcaaatc cagccgttac
     3241 ttttggcaca gctattgcgg ctttttactt gcccgtggtg attatgaccg tcttgtatat
     3301 ccatatctca ctggctagtc ggagccgggt gcataagcac cggcctgaag ggccaaagga
     3361 gaagaaagca aagacattgg ccttcctcaa aagccccttg atgaagcaat ccgtgaaaaa
     3421 acctcccccg ggagaggctg cccgcgaaga gcttcggaac ggcaagttgg aagaagcacc
     3481 acctcccgct ctgccaccac ccccacggcc cgtggctgat aaggacacta gcaacgagag
     3541 cagcagcggg agcgccacac agaacactaa agagcggccg gccacagagc tctcaacaac
     3601 cgaggctacc acgccagcta tgccagcacc gccactccaa cctcgggctc ttaaccctgc
     3661 aagtcggtgg agcaagattc agattgtcac caagcagaca ggcaatgaat gtgttacggc
     3721 tattgaaatc gtgccagcta cccctgccgg gatgcgaccc gctgctaatg tggccagaaa
     3781 gttcgccagc atagcacgca accaagtccg gaaaaaaaga caaatggccg cacgggagag
     3841 aaaggtgaca cgcacgattt tcgctattct tctggccttc atactgactt ggacccctta
     3901 caatgtgatg gtcctggtaa acaccttttg ccagtcatgt atccccgaca cagtgtggag
     3961 catcggatat tggctgtgct acgtgaacag caccataaac ccggcttgct atgccctctg
     4021 taacgctacg ttcaaaaaga cgtttcgcca tctgttgctt tgtcaatacc ggaacattgg
     4081 tactgctcgc atcgataccg gtggacgcac cggtgaattc gatgaggcca gcaccaccgt
     4141 gtctaagacc gagacttctc aggtggcccc tgcctaactc gagtctagag ggcccgttta
     4201 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     4261 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     4321 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     4381 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     4441 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     4501 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4561 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4621 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4681 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     4741 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4801 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4861 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     4921 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     4981 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     5041 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     5101 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     5161 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     5221 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     5281 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     5341 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     5401 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     5461 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     5521 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     5581 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     5641 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     5701 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     5761 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     5821 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     5881 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     5941 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     6001 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     6061 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     6121 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     6181 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     6241 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     6301 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     6361 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     6421 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     6481 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     6541 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     6601 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6661 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     6721 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6781 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6841 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6901 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6961 cgtttccccc tggaagc
//
