LOCUS       sequence-430823-        6587 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 6587)
  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 6587)
  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: GPR21-DuET
FEATURES             Location/Qualifiers
     source          1..6587
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(60..851,852..920))
                     /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     683..702
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(921..1025)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1100..1119)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1291..1670
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1671..1874
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1825..1845
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1919..1938
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1919..1937
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3234..3251)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3240..3464
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3510..3938
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3597..3616)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3807..3828
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3947..3967)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3952..4281
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4132..4267
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4194..4213
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4348..5142
                     /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(4402..4421)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5012..5031
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5316..5437
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5353..5372)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5407..5426
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5486..5502)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5486..5502)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5499..5521)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5510..5526
                     /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(5534..5540,5541..5558,5559..5564))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5579..5600
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5717..5734)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5888..6476)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5968..5987)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
       61 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
      121 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
      181 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
      241 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
      301 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
      361 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
      421 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
      481 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
      541 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
      601 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
      661 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
      721 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
      781 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
      841 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
      901 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
      961 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     1021 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     1081 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     1141 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     1201 ggcaaggctt gaccgacatt tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     1261 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
     1321 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     1381 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     1441 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     1501 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     1561 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     1621 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     1681 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     1741 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     1801 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     1861 atataagcag agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta
     1921 atacgactca ctatagggag acccaagctg gctagcgttt aaacttaagc ttagcgccac
     1981 catgaagacg atcatcgccc tgagctacat cttctgcctg gtattcgccg actacaagga
     2041 cgatgatgac gccagcatcg atatgaactc caccctcgat ggtaaccaaa gctcccaccc
     2101 attctgcctg ctggccttcg gatatcttga gaccgttaac ttttgccttc tggaagtcct
     2161 gatcattgtg tttcttaccg tgctcataat tagcgggaat ataatcgtga tcttcgtgtt
     2221 tcactgtgca cctcttttga accaccatac aacttcctac ttcatacaaa caatggcgta
     2281 tgccgatctt tttgtgggtg tatcttgtgt ggtgccatca ctctcacttc tgcaccatcc
     2341 cctgccagtg gaggagtcac ttacttgcca gatcttcgga tttgtggtgt ccgttctcaa
     2401 gtctgtgagt atggccagtt tggcctgtat ctctatagac cgatacatcg ctatcacaaa
     2461 gccactcacg tacaatacac tggtaacacc atggcgactt cgcttgtgta tcttcttgat
     2521 ttggctgtac tctacattgg tgttccttcc aagctttttt cactggggga agcccggata
     2581 tcatggagat gtgttccagt ggtgtgcaga gtcatggcat accgactcat atttcacatt
     2641 gtttattgtc atgatgttgt acgctcctgc agctcttatt gtgtgcttta cgtacttcaa
     2701 catctttcgg atctgccagc agcacactaa agacatctct gagcgccagg ctcgcttcag
     2761 ctcccaaagc ggtgaaacag gcgaggtgca agcctgccct gacaaacgct acgcaatggt
     2821 gctgtttcga atcaccagcg tcttttacat tttgtggctg ccatacataa tttatttcct
     2881 tctggagtcc agcaccgggc actccaaccg cttcgcaagc tttctgacga catggcttgc
     2941 catttctaac agcttctgca actgtgtgat ttatagcctg tcaaattcag tattccagcg
     3001 gggtcttaag cggctcagtg gcgcaatgtg taccagctgt gccagtcaga ccaccgcaaa
     3061 tgatccctac accgtgagaa gcaagggacc actgaacgga tgtcatatca tcgataccgg
     3121 tggacgcacc ggtgaattcg atgaggccag caccaccgtg tctaagaccg agacttctca
     3181 ggtggcccct gcctaactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     3241 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3301 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3361 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3421 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3481 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3541 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3601 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3661 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3721 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3781 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3841 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3901 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3961 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4021 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4081 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4141 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4201 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     4261 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     4321 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     4381 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4441 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4501 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4561 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4621 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4681 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4741 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4801 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4861 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4921 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4981 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     5041 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     5101 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     5161 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     5221 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5281 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5341 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5401 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5461 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5521 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5581 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5641 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5701 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5761 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5821 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5881 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5941 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6001 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6061 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6121 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6181 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6241 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6301 ggtggcctaa ctacggctac actagaagaa cggtatttgg tatctgcgct ctgctgaagc
     6361 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6421 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6481 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6541 ttttggtcat gagattatca aaaaggatct tcacctagat cctttta
//
