LOCUS       sequence-430753-        6653 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 6653)
  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 6653)
  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: AVPR2-DuET
FEATURES             Location/Qualifiers
     source          1..6653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(51..67)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(51..67)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(64..86)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    75..91
                     /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(99..105,106..123,124..129))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    144..165
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(282..299)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(453..1041)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(533..552)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1212..2003,2004..2072))
                     /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     1835..1854
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2073..2177)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2252..2271)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2443..2822
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2823..3026
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2977..2997
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3071..3090
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3071..3089
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4452..4469)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4458..4682
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4728..5156
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4815..4834)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5025..5046
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5165..5185)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5170..5499
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5350..5485
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5412..5431
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5566..6360
                     /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(5620..5639)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6230..6249
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    join(6534..6653,1..2)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6571..6590)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6625..6644
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
       61 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
      121 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
      181 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
      241 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
      301 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
      361 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
      421 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
      481 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
      541 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
      601 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
      661 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
      721 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
      781 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
      841 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacggta
      901 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
      961 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     1021 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     1081 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     1141 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     1201 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     1261 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     1321 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     1381 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     1441 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     1501 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     1561 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     1621 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     1681 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     1741 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     1801 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     1861 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     1921 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     1981 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     2041 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     2101 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     2161 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     2221 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     2281 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     2341 aagctacaac aaggcaaggc ttgaccgaca tttgcatgaa gaatctgctt agggttaggc
     2401 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     2461 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     2521 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     2581 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     2641 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     2701 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     2761 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     2821 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     2881 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     2941 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     3001 ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc
     3061 ttatcgaaat taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa
     3121 gcttagcgcc accatgaaga cgatcatcgc cctgagctac atcttctgcc tggtattcgc
     3181 cgactacaag gacgatgatg acgccagcat cgatatgctc atggcctcta ccacaagcgc
     3241 tgtgcccgga catccatctc ttccaagtct tccctccaac agctcacagg agaggcctct
     3301 ggatacccgg gacccacttc ttgcaagagc ggagctcgcc ttgctctcca tcgttttcgt
     3361 tgccgtcgcg ttgagcaatg gtctggtgct ggccgccctg gcgcgacgag gccgccgagg
     3421 ccactgggcg cctattcatg tctttatcgg ccatctctgt cttgcagacc tggccgtggc
     3481 gctgtttcag gtgctccctc aactggcttg gaaagccacc gatagattcc gggggccaga
     3541 cgcactgtgt cgggccgtga aatacctgca gatggtggga atgtatgcca gttcatatat
     3601 gatacttgct atgaccttgg acagacacag ggctatctgt cgccccatgc tggcgtatag
     3661 gcacggttcc ggagcccatt ggaacaggcc agttctggtt gcgtgggctt tttccctgct
     3721 tctgagcttg ccccagttgt ttattttcgc acagagaaat gtggagggcg gatctggcgt
     3781 gaccgattgc tgggcttgct tcgctgagcc atggggcaga aggacctacg tgacgtggat
     3841 tgctctcatg gtgttcgttg ctcccaccct ggggatcgcc gcatgccagg tcctgatatt
     3901 cagagaaata cacgcctcct tggttcctgg gccaagcgag cgccctggtg gaagaagacg
     3961 gggtcgaagg actggctctc caggcgaagg cgcccacgta tcagctgcgg tagctaagac
     4021 cgtacgcatg acactggtca tcgtcgtcgt ttacgttctt tgctgggccc cgtttttttt
     4081 ggtgcagctc tgggcggcct gggaccccga agcacccctg gaaggtgctc cattcgttct
     4141 gctcatgctt ctcgcttcac tgaatagctg taccaatcct tggatttacg cctctttctc
     4201 ctccagtgtt agctccgagc tgcggagtct tctctgttgc gcccggggca ggaccccccc
     4261 aagcctgggt ccacaggacg aatcctgcac taccgcctcc agttccctgg ccaaagacac
     4321 ctcttctatc gataccggtg gacgcaccgg tgaattcgat gaggccagca ccaccgtgtc
     4381 taagaccgag acttctcagg tggcccctgc ctaactcgag tctagagggc ccgtttaaac
     4441 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     4501 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     4561 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     4621 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     4681 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     4741 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4801 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4861 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4921 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     4981 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5041 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     5101 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     5161 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     5221 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     5281 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     5341 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     5401 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     5461 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     5521 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     5581 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     5641 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     5701 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     5761 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     5821 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     5881 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     5941 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     6001 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     6061 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     6121 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     6181 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     6241 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     6301 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     6361 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     6421 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     6481 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     6541 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     6601 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tct
//
