LOCUS       sequence-430845-        6578 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 6578)
  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 6578)
  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: HCA1-DuET
FEATURES             Location/Qualifiers
     source          1..6578
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     256..275
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(494..598)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(673..692)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        864..1243
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1244..1447
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1398..1418
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1492..1511
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1492..1510
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2798..2815)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2804..3028
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3074..3502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3161..3180)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3371..3392
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3511..3531)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3516..3845
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3696..3831
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3758..3777
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3912..4706
                     /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(3966..3985)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4576..4595
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4880..5001
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4917..4936)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4971..4990
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5050..5066)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5050..5066)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5063..5085)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5074..5090
                     /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(5098..5104,5105..5122,5123..5128))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5143..5164
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5281..5298)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5452..6040)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5532..5551)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6211..6578,1..424,425..493))
                     /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 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
       61 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
      121 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
      181 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
      241 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
      301 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
      361 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
      421 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
      481 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
      541 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
      601 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
      661 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
      721 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
      781 cttgaccgac atttgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
      841 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
      901 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      961 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     1021 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     1081 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     1141 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     1201 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     1261 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     1321 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     1381 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     1441 cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac
     1501 tcactatagg gagacccaag ctggctagcg tttaaactta agcttagcgc caccatgaag
     1561 acgatcatcg ccctgagcta catcttctgc ctggtattcg ccgactacaa ggacgatgat
     1621 gacgccagca tcgatatgta taatggctcc tgttgccgga ttgaaggcga cactatctct
     1681 caagtaatgc cccccctcct gatagtggcc ttcgtcctgg gcgctcttgg taatggggtt
     1741 gcactctgtg gtttctgttt ccacatgaaa acctggaagc cctcaacagt gtacctcttt
     1801 aaccttgccg tggcagactt cctgcttatg atttgcctgc cctttaggac cgactattat
     1861 ctgcgacgcc gccattgggc tttcggcgac atcccctgtc gggttggtct gtttactctg
     1921 gctatgaatc gggccggcag tatcgtcttt ctcactgtgg tcgctgccga cagatacttc
     1981 aaggtagtgc acccccacca cgcagtgaac acaatctcca ctagagttgc agctggaatt
     2041 gtgtgcaccc tgtgggctct ggtaatcctg ggcacagtat acctgctcct ggagaatcat
     2101 ttgtgcgtgc aggagactgc tgtgtcatgt gaatctttta ttatggagtc cgcaaacggg
     2161 tggcatgata tcatgtttca actggagttc tttatgcccc ttggcatcat tctgttttgc
     2221 tcattcaaga tcgtttggtc tctccggcgc cggcagcagc tggcccggca agctcggatg
     2281 aaaaaggcca cgcgctttat catggttgtg gctatcgtct tcatcacctg ctaccttcct
     2341 tccgtgtccg caagactgta ttttctgtgg accgtcccca gcagcgcttg cgatcccagt
     2401 gtccacggcg ccctccacat caccttgagc tttacgtaca tgaactctat gctggacccc
     2461 ctggtgtact attttagctc tccctccttc ccgaaattct ataataaact taagatctgc
     2521 agcctcaaac caaagcaacc aggccattcc aagactcagc gccctgaaga gatgcccatt
     2581 agcaaccttg gtagacggag ctgcatctcc gtcgcgaact catttcagtc tcagtccgac
     2641 ggacagtggg acccacacat tgttgagtgg cacatcgata ccggtggacg caccggtgaa
     2701 ttcgatgagg ccagcaccac cgtgtctaag accgagactt ctcaggtggc ccctgcctaa
     2761 ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     2821 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2881 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2941 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3001 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3061 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3121 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3181 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3241 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3301 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3361 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3421 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3481 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3541 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3601 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3661 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3721 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3781 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3841 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     3901 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3961 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4021 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4081 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4141 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4201 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4261 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4321 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4381 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4441 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4501 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4561 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4621 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4681 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4741 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4801 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4861 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     4921 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4981 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     5041 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5101 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5161 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5221 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5281 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5341 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5401 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5461 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5521 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5581 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5641 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5701 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5761 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5821 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5881 ctacactaga agaacggtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5941 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6001 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6061 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6121 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6181 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6241 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6301 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6361 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6421 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6481 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6541 gtcacgctcg tcgtttggta tggcttcatt cagctccg
//
