LOCUS       sequence-430840-        6749 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 6749)
  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 6749)
  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: GPRC5B-DuET
FEATURES             Location/Qualifiers
     source          1..6749
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2..19)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(173..761)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(253..272)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(932..1723,1724..1792))
                     /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     1555..1574
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1793..1897)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1972..1991)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2163..2542
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2543..2746
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2697..2717
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2791..2810
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2791..2809
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4268..4285)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4274..4498
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4544..4972
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4631..4650)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4841..4862
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4981..5001)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4986..5315
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5166..5301
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5228..5247
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5382..6176
                     /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(5436..5455)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6046..6065
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6350..6471
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6387..6406)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6441..6460
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6520..6536)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6520..6536)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6533..6555)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6544..6560
                     /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(6568..6574,6575..6592,6593..6598))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6613..6634
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
       61 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
      121 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
      181 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
      241 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
      301 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
      361 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
      421 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
      481 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
      541 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
      601 gctacactag aagaacggta tttggtatct gcgctctgct gaagccagtt accttcggaa
      661 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
      721 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
      781 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
      841 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
      901 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
      961 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     1021 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     1081 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     1141 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     1201 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     1261 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     1321 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     1381 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     1441 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     1501 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     1561 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     1621 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     1681 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     1741 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     1801 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     1861 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     1921 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     1981 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     2041 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca tttgcatgaa
     2101 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
     2161 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     2221 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     2281 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     2341 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2401 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     2461 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     2521 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     2581 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     2641 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     2701 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
     2761 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
     2821 tggctagcgt ttaaacttaa gcttagcgcc accatgaaga cgatcatcgc cctgagctac
     2881 atcttctgcc tggtattcgc cgactacaag gacgatgatg acgccagcat cgatatgttc
     2941 gtggcttctg aacggaaaat gcgagcacat caggtactga cttttctcct cctgtttgtg
     3001 atcacctctg tcgcttccga gaacgctagc acaagtaggg gctgtggact ggatctcttg
     3061 cctcagtatg tcagtttgtg tgatctggat gcgatctggg gaatcgttgt ggaggctgta
     3121 gccggtgcag gagctcttat aacccttttg ctgatgctta tcctcttggt taggcttccc
     3181 ttcatcaaag aaaaagagaa aaaatcccca gttggactgc acttcttgtt cctccttggc
     3241 accttggggc tgttcggact cacttttgcc ttcatcatcc aggaagatga aaccatctgt
     3301 agtgttcgga gattcctgtg gggagtgttg tttgcactgt gcttttcatg cttgcttagt
     3361 caggcatgga gggtgagaag gttggtgagg catggcactg gccctgccgg gtggcaactg
     3421 gttgggctcg ccctgtgcct gatgctggta caggtcatca tcgctgttga atggctggtc
     3481 ctgactgtgc tgcgggacac gcgccccgct tgcgcatacg agccgatgga cttcgtcatg
     3541 gcccttatat acgatatggt actgctggtg gtaaccctcg ggctggcact gtttactctc
     3601 tgcgggaaat tcaagcgatg gaaactgaat ggggcttttc tgctgatcac agctttcttg
     3661 agcgtgctca tctgggtggc ctggatgacg atgtacctct tcggcaacgt gaagctccag
     3721 cagggcgacg cgtggaatga cccgaccctg gccatcacgc tggcagctag cggctgggtg
     3781 ttcgtgatat ttcatgccat ccccgaaatt cactgtaccc ttcttcccgc cttgcaagag
     3841 aataccccca actatttcga cacaagccaa ccgcgcatga gagaaactgc ttttgaggag
     3901 gacgtccagc tcccaagagc ttacatggag aacaaggctt tctctatgga cgaacacaac
     3961 gctgctttga gaacagctgg atttcctaat ggctcactgg gtaaaaggcc gagtggatcc
     4021 ctcggcaaga ggccatctgc cccatttcga agtaatgtct atcagccaac agagatggct
     4081 gttgtcctca atgggggtac tatacccaca gccccaccat cccatacagg aaggcacctg
     4141 tggatcgata ccggtggacg caccggtgaa ttcgatgagg ccagcaccac cgtgtctaag
     4201 accgagactt ctcaggtggc ccctgcctaa ctcgagtcta gagggcccgt ttaaacccgc
     4261 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4321 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4381 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4441 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     4501 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     4561 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4621 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4681 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4741 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4801 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4861 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     4921 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     4981 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     5041 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     5101 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     5161 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     5221 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     5281 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     5341 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     5401 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     5461 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     5521 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     5581 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     5641 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     5701 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     5761 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     5821 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     5881 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     5941 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     6001 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     6061 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     6121 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     6181 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     6241 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     6301 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     6361 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     6421 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     6481 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     6541 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     6601 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     6661 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     6721 cggtttgcgt attgggcgct cttccgctt
//
