LOCUS       sequence-430877-        6692 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 6692)
  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 6692)
  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: NPY1R-DuET
FEATURES             Location/Qualifiers
     source          1..6692
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     341..360
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     594..611
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    728..749
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(764..769,770..787,788..794)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    802..818
                     /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     807..829
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     826..842
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     826..842
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    891..1012
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(902..921)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     956..975
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1186..1980)
                     /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(1297..1316)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1907..1926
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2047..2376)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2061..2196
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2115..2134)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2361..2381
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2390..2818)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2500..2521)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2712..2731
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2864..3088
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3077..3094
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4495..4514)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4496..4514)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4559..4762)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4588..4608)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4763..5142
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5314..5333
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5408..5512
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5513..5581,5582..6373)
                     /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(5731..5750)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6544..6692,1..440)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
       61 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
      121 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
      181 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
      241 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
      301 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
      361 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      421 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      481 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
      541 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
      601 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      661 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      721 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      781 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
      841 accatgatta cgccaagctc tagctagagg tcgacggtat acagacatga taagatacat
      901 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
      961 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttggggtggg
     1021 cgaagaactc cagcatgaga tccccgcgct ggaggatcat ccagccggcg tcccggaaaa
     1081 cgattccgaa gcccaacctt tcatagaagg cggcggtgga atcgaaatct cgtgatggca
     1141 ggttgggcgt cgcttggtcg gtcatttcga accccagagt cccgctcaga agaactcgtc
     1201 aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag
     1261 gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat
     1321 gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc
     1381 attttccacc atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc
     1441 gtcgggcatg cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc
     1501 ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat
     1561 gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg
     1621 cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc
     1681 ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag
     1741 cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg
     1801 cagttcattc agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc
     1861 tgacagccgg aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc
     1921 gaatagcctc tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat
     1981 gcgaaacgat cctcatcctg tctcttgatc agatccgaaa atggatatac aagctcccgg
     2041 gagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc
     2101 agaggcagag gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg
     2161 agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat
     2221 ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga
     2281 ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
     2341 ggagcctggg gactttccac accctaactg acacacattc cacagaatta attcgcgtta
     2401 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     2461 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     2521 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     2581 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     2641 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     2701 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     2761 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg
     2821 ggataccccc tagagcccca gctggttctt tccgcctcag aagccataga gcccaccgca
     2881 tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca
     2941 ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag
     3001 gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca
     3061 acagatggct ggcaactaga aggcacagtc gaggctgatc agcgggttta aacgggccct
     3121 ctagactcga gttaggcagg ggccacctga gaagtctcgg tcttagacac ggtggtgctg
     3181 gcctcatcga attcaccggt gcgtccaccg gtatcgataa ttttttcatt gtcgtcgttg
     3241 ttgtttattt tcttgaaagc cacggggctg gcctgcttca gactcgtttt agaaacgtcc
     3301 gtgtgcattg tactcattgc tattgtctcg taatcgtcat cccgggagcg gaaatcgcaa
     3361 aagttgaaaa aaaactgcag gtcccgctgg aagttcttgt tcaaaaatcc gtaaaagatc
     3421 gggttcacgc atgtactgat catggctgtc aaatggcaga gcaggaacaa caggttatgg
     3481 ttacaggttg cgatgatctg atgattccaa tcgaacacag tgttaaagat tgtaagaggc
     3541 agccaacaaa ctgcgaacgc aaccacaatg gacagcagca taatgttgat gcgcttagtc
     3601 tctgagctgc ggtatttgtt atctctcatc ttgtccatca tgttattcct gcgtttgagt
     3661 ctgatataga tcttgaagta gcatataaag ataaaacaga gggggccgaa gtactgaaga
     3721 accaacagca gtgttgtata actcaatctg tgggagtcac tggggaactg gtcgaaacac
     3781 acatacttat ccttataagc gtcaagagtt acattttgaa aaggctcgtc tgtcatcacc
     3841 tggtaaatga ggaagggcag ggaactggcc accgccaaca cccagattac cgctattccc
     3901 acgtaagcat ggcggttatt aggtcgccac cctcggggat tgataatcag ctgatgtctt
     3961 tcgacggcaa tcaggacgag agagaatatg gacaccgtaa tggagacaca ctgcacgaaa
     4021 gggttcaatt tgcacatggc ctcgccaaac acccagtggt ccatcagagt gtacacaaag
     4081 gtaaatggaa gacacataat tgctacgagg agatcgctaa atgacagatt gactatcaaa
     4141 atgttcgtca cattcctcat ttctttttgc ttaagaatga taatgatgag agcgaggttt
     4201 cctgaaacgc ccagtatgat gacggctccg taagccaggg cgagggtgaa aatcatagcc
     4261 agtggcaggt gacaatcgtc attctcaaac gcaagcagct gtgcgttctt ctcactgaaa
     4321 ttggaatgga cactatgatt ctccacctga gaaaaaagtg ttgaattcat atcgatgctg
     4381 gcgtcatcat cgtccttgta gtcggcgaat accaggcaga agatgtagct cagggcgatg
     4441 atcgtcttca tggtggcgct aagcttaagt ttaaacgcta gccagcttgg gtctccctat
     4501 agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt agccagagag
     4561 ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc aatggggcgg
     4621 agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa ctcccattga
     4681 cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga
     4741 tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag atgtactgcc
     4801 aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc catttaccgt
     4861 cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg
     4921 cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta
     4981 tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg
     5041 ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga actaatgacc
     5101 ccgtaattga ttactattaa taactagtca ataatcaatg tcaacgcgta tatctggccc
     5161 gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg caaatgtcgg
     5221 tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg acctccaaca
     5281 cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt gtactgagag
     5341 tgcaccatag gggatcggga gatctcccga tccgtcgacg tcaggtggca cttttcgggg
     5401 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5461 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5521 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5581 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5641 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5701 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5761 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5821 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5881 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5941 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6001 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6061 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6121 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6181 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6241 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6301 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6361 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6421 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6481 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6541 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6601 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6661 cttcagcaga gcgcagatac caaataccgt tc
//
