LOCUS       sequence-430846-        6629 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 6629)
  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 6629)
  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: HCA2-DuET
FEATURES             Location/Qualifiers
     source          1..6629
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     359..378
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(597..701)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(776..795)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        967..1346
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1347..1550
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1501..1521
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1595..1614
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1595..1613
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2952..2969)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2958..3182
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3228..3656
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3315..3334)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3525..3546
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3665..3685)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3670..3999
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3850..3985
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3912..3931
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4066..4860
                     /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(4120..4139)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4730..4749
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5034..5155
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5071..5090)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5125..5144
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5204..5220)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5204..5220)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5217..5239)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5228..5244
                     /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(5252..5258,5259..5276,5277..5282))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5297..5318
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5435..5452)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5606..6194)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5686..5705)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6365..6629,1..527,528..596))
                     /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 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
       61 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
      121 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
      181 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
      241 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
      301 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
      361 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
      421 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
      481 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
      541 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
      601 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
      661 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
      721 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
      781 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      841 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacatttgca
      901 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      961 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     1021 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     1081 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     1141 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     1201 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     1261 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     1321 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     1381 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     1441 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     1501 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
     1561 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
     1621 aagctggcta gcgtttaaac ttaagcttag cgccaccatg aagacgatca tcgccctgag
     1681 ctacatcttc tgcctggtat tcgccgacta caaggacgat gatgacgcca gcatcgatat
     1741 gaaccggcac cacctgcagg atcacttcct ggaaatagac aaaaagaact gctgcgtttt
     1801 tagagacgac tttatcgtca aagtactgcc cccagtgctc ggcctggagt tcatcttcgg
     1861 acttctgggg aatggcctcg ccctctggat tttttgcttc caccttaagt cctggaagag
     1921 tagccgaatc ttcctgttca atctcgccgt agcggatttt ctgctcatca tttgcctgcc
     1981 tttcctgatg gacaattatg tgcgccggtg ggattggaaa ttcggtgata ttccttgtag
     2041 attgatgctc ttcatgctgg ctatgaaccg gcaaggaagc ataatctttc ttaccgttgt
     2101 ggccgtggac agatacttcc gagtggtgca tccacatcat gccctcaata agatttccaa
     2161 tcgaacggcc gctataatta gctgtcttct gtggggaatc accatcggcc tcaccgtcca
     2221 cctcctcaag aagaagatgc cgatccagaa cggtggggcc aacctgtgta gttcctttag
     2281 tatatgccat actttccagt ggcatgaggc tatgttcctg ctggagttct tcctgccact
     2341 tggcatcatc ttgttctgct ccgctcggat aatatggagc ctgagacaga gacagatgga
     2401 tcggcatgcg aaaataaagc gggccattac gttcatcatg gtcgtggcta ttgtgtttgt
     2461 gatttgtttc ctcccctctg tggtggtgag aataagaatt ttttggcttc tccacacgag
     2521 cggcacccaa aattgcgagg tttacaggag cgtggatctt gcctttttca tcactctgag
     2581 cttcacatac atgaattcaa tgctcgatcc agtggtttac tacttcagca gtccttcatt
     2641 cccgaacttt ttctctacgc tcatcaatcg gtgcctgcag cgcaaaatga ctggcgagcc
     2701 cgacaacaac agaagcacct ccgtggaact caccggcgac cctaacaaga cccggggtgc
     2761 ccccgaggct ctgatggcga actctggtga gccctggtcc cctagctatc tgggcccaac
     2821 gagccccatc gataccggtg gacgcaccgg tgaattcgat gaggccagca ccaccgtgtc
     2881 taagaccgag acttctcagg tggcccctgc ctaactcgag tctagagggc ccgtttaaac
     2941 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3001 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3061 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3121 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3181 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     3241 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3301 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3361 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3421 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3481 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3541 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3601 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     3661 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3721 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3781 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3841 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3901 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     3961 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     4021 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4081 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4141 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4201 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4261 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4321 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4381 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4441 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4501 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4561 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     4621 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4681 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4741 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4801 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4861 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4921 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4981 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     5041 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5101 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5161 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     5221 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5281 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5341 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5401 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5461 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5521 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5581 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5641 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5701 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5761 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5821 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5881 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5941 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6001 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaacg gtatttggta
     6061 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6121 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6181 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6241 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6301 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6361 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6421 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6481 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6541 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6601 tccagtctat taattgttgc cgggaagct
//
