LOCUS       sequence-430915-        6539 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 6539)
  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 6539)
  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: NPBW2-DuET
FEATURES             Location/Qualifiers
     source          1..6539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     404..423
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(642..746)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(821..840)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1012..1391
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1392..1595
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1546..1566
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1640..1659
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1640..1658
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2907..2924)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2913..3137
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3183..3611
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3270..3289)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3480..3501
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3620..3640)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3625..3954
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3805..3940
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3867..3886
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4021..4815
                     /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(4075..4094)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4685..4704
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4989..5110
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5026..5045)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5080..5099
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5159..5175)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5159..5175)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5172..5194)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5183..5199
                     /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(5207..5213,5214..5231,5232..5237))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5252..5273
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5390..5407)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5561..6149)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5641..5660)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6320..6539,1..572,573..641))
                     /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 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
       61 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
      121 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
      181 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
      241 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
      301 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
      361 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
      421 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
      481 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
      541 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
      601 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
      661 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
      721 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
      781 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
      841 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
      901 gcaaaattta agctacaaca aggcaaggct tgaccgacat ttgcatgaag aatctgctta
      961 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
     1021 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     1081 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     1141 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     1201 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     1261 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     1321 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     1381 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
     1441 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
     1501 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
     1561 ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg
     1621 cttactggct tatcgaaatt aatacgactc actataggga gacccaagct ggctagcgtt
     1681 taaacttaag cttagcggcc gcgccaccat gaagacgatc atcgccctga gctacatctt
     1741 ctgcctggta ttcgccgact acaaggacga tgatgacgcc agcatcgata tgcaggcagc
     1801 gggccaccca gaacccctcg atagccgagg gtccttttcc ctgcccacga tgggcgccaa
     1861 tgtatctcag gacaacggga ctggccataa cgctactttc tccgaaccac tcccatttct
     1921 gtatgtcctc ctgccagccg tttacagcgg gatctgcgca gtgggattga cgggtaacac
     1981 agcagtgatc ctcgtcatcc tgcgagcacc caaaatgaag acggtgacca atgtgttcat
     2041 ccttaatttg gctgtggccg acggactgtt tactctggtc ctgcccgtga acatagctga
     2101 acacttgttg cagtactggc cgtttgggga actgttgtgt aaactggtgc tcgcagtcga
     2161 ccattataat atattctctt ccatttactt tcttgctgtg atgagcgttg accgctatct
     2221 ggtcgtactt gcgaccgtaa gatctcggca catgccctgg cgcacgtata ggggcgcaaa
     2281 ggtggcctct ctgtgcgtct ggctcggggt gaccgtgctg gtgcttccct tcttcagttt
     2341 tgccggtgtg tactccaacg aacttcaagt gccctcctgt ggcctgtcat tcccatggcc
     2401 agaacaagtg tggttcaaag caagcagagt ctacacgttg gtactgggat ttgtcctccc
     2461 agtgtgcacc atctgcgtgc tgtacacaga tctgctgcga aggctcagag ctgttagact
     2521 caggagtggc gccaaagcac ttggcaaagc taggcggaaa gtgactgtgt tggtgctcgt
     2581 cgtgttggct gtgtgtctgc tttgctggac cccctttcac cttgcttctg tagtagctct
     2641 cacaaccgac ctcccccaaa cacccctggt gatttccatg agctatgtta tcacctcact
     2701 ttcctacgca aacagttgtc tgaatccgtt tctgtacgct ttcctggatg ataacttccg
     2761 gaaaaatttc aggtcaatcc tccgctgtat cgataccggt ggacgcaccg gtgaattcga
     2821 tgaggccagc accaccgtgt ctaagaccga gacttctcag gtggcccctg cctaatctag
     2881 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2941 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     3001 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     3061 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     3121 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     3181 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3241 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3301 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3361 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3421 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3481 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3541 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3601 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3661 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3721 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3781 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3841 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     3901 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3961 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     4021 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     4081 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4141 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4201 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4261 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4321 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4381 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4441 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4501 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     4561 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4621 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4681 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4741 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4801 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4861 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4921 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4981 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5041 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5101 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     5161 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5221 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5281 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5341 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5401 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5461 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5521 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5581 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5641 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5701 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5761 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5821 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5881 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5941 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6001 gaacggtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6061 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6121 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6181 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6241 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6301 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6361 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6421 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6481 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgca
//
