LOCUS       sequence-430810-        6542 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 6542)
  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 6542)
  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: GPR12-DuET
FEATURES             Location/Qualifiers
     source          1..6542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     594..613
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(832..936)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1011..1030)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1202..1581
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1582..1785
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1736..1756
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1830..1849
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1830..1848
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3100..3117)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3106..3330
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3376..3804
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3463..3482)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3673..3694
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3813..3833)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3818..4147
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3998..4133
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4060..4079
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4214..5008
                     /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(4268..4287)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4878..4897
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5182..5303
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5219..5238)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5273..5292
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5352..5368)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5352..5368)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5365..5387)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5376..5392
                     /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(5400..5406,5407..5424,5425..5430))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5445..5466
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5583..5600)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5754..6342)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5834..5853)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6513..6542,1..762,763..831))
                     /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 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
       61 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
      121 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
      181 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
      241 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
      301 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
      361 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
      421 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
      481 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
      541 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
      601 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
      661 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
      721 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
      781 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
      841 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
      901 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
      961 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     1021 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     1081 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacat ttgcatgaag
     1141 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     1201 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
     1261 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     1321 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     1381 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     1441 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     1501 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     1561 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     1621 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     1681 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     1741 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga
     1801 gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct
     1861 ggctagcgtt taaacttaag cttagcgcca ccatgaagac gatcatcgcc ctgagctaca
     1921 tcttctgcct ggtattcgcc gactacaagg acgatgatga cgccagcatc gatatgaacg
     1981 aagatctgaa agtgaatctc tccggtcttc ccagggacta cctcgatgcg gctgctgccg
     2041 agaacatatc cgcagccgtg tcatctagag ttcctgcggt tgagcccgag ccagagcttg
     2101 tggtcaatcc ctgggacatc gtcctgtgta catccgggac cctgatcagt tgcgagaacg
     2161 cgattgtcgt cctgatcata tttcacaatc cgtccctccg ggcgcctatg ttcctcctca
     2221 ttggttccct cgcgttggcc gatctgctgg ccggcatcgg acttataact aattttgtct
     2281 ttgcgtacct cctgcaatct gaggccacta agcttgttac tatcggactg atcgtggctt
     2341 ctttctccgc aagtgtctgt tccctcctcg ccatcaccgt ggatcgctat ttgagtcttt
     2401 actatgccct cacctaccat tcagagagaa ctgttacctt tacgtatgtg atgctggtta
     2461 tgctttgggg gacctccatc tgcttgggac tcctgcctgt tatgggttgg aactgtctga
     2521 gagacgagtc cacttgcagt gtggtgaggc cgctgactaa gaataacgcg gctatactct
     2581 ctgtgagttt ccttttcatg ttcgcattga tgttgcaatt gtacatccag atctgcaaga
     2641 tcgtgatgcg ccacgcgcac caaatcgcac tgcagcatca ttttctggca acaagtcact
     2701 atgtgactac caggaaaggg gttagcactc ttgccatcat cttgggtact ttcgctgcct
     2761 gttggatgcc gttcactctg tattccctga tcgcggacta cacatatcca tctatttaca
     2821 catacgccac cctgctccca gccacctaca acagcatcat aaaccccgtc atctatgcct
     2881 tcaggaacca ggagatccag aaagctctgt gtctgatctg ctgcggctgt attcctagta
     2941 gccttgcaca acgagccaga agcccaagcg acgtgatcga taccggtgga cgcaccggtg
     3001 aattcgatga ggccagcacc accgtgtcta agaccgagac ttctcaggtg gcccctgcct
     3061 aactcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     3121 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3181 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3241 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     3301 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     3361 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3421 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3481 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3541 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3601 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3661 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3721 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3781 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     3841 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3901 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3961 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4021 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4081 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     4141 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     4201 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     4261 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     4321 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4381 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4441 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4501 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4561 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4621 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4681 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4741 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4801 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4861 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4921 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4981 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     5041 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     5101 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5161 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     5221 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5281 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     5341 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5401 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5461 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5521 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5581 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5641 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5701 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5761 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5821 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5881 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5941 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6001 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6061 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6121 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6181 ggctacacta gaagaacggt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6241 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6301 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6361 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6421 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6481 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6541 at
//
