LOCUS       sequence-430748-        6779 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 6779)
  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 6779)
  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: ADRB2-DuET
FEATURES             Location/Qualifiers
     source          1..6779
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(557..576)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(558..576)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(621..824)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(650..670)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        825..1204
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1376..1395
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1470..1574
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1575..1643,1644..2435)
                     /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     complement(1793..1812)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2606..3194
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3095..3114
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3348..3365
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3482..3503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3518..3523,3524..3541,3542..3548)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3556..3572
                     /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)."
     primer_bind     3561..3583
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3580..3596
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3580..3596
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3645..3766
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3656..3675)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3710..3729
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3940..4734)
                     /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(4051..4070)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4661..4680
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4801..5130)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4815..4950
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4869..4888)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5115..5135
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5144..5572)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5254..5275)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5466..5485
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5618..5842
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5831..5848
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 cagagactgg tatttaaagg gactcgtaat cgcgaagtac ctatcaacgg caataacaca
       61 cagagtttcg attgacgctg tgacgcagag cacgtcaatt gaagtccaga attcgcacca
      121 aaaattacca aaagtccaca tcttcatcag gatatgagct gcgccaaacg gtacaaccgc
      181 aaggcccatg accaagtcgg cacaggcgag ggaagtaata aagtaattgg taaccgtctg
      241 aagccgctcg aactttgcaa tagcagtaat gacgaggacg ttgccgaaca caattgccag
      301 gacgatcagt gacatcacaa tacccatgcc tacaacccag acctcgtctc tctgctgagt
      361 cacatcgtgg tcgggagcgt ggctccgatt aggggcgagg agaaaagctg atccattgcc
      421 aggttgcccc atatcgatgc tggcgtcatc atcgtccttg tagtcggcga ataccaggca
      481 gaagatgtag ctcagggcga tgatcgtctt catggtggcg ctaagcttaa gtttaaacgc
      541 tagccagctt gggtctccct atagtgagtc gtattaattt cgataagcca gtaagcagtg
      601 ggttctctag ttagccagag agctctgctt atatagacct cccaccgtac acgcctaccg
      661 cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg ttgattttgg
      721 tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca
      781 aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg
      841 actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa
      901 tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac
      961 acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg
     1021 aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg
     1081 tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata
     1141 tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt caataatcaa
     1201 tgtcaacgcg tatatctggc ccgtacatcg cgaagcagcg caaaacgcct aaccctaagc
     1261 agattcttca tgcaaatgtc ggtcaagcct tgccttgttg tagcttaaat tttgctcgcg
     1321 cactactcag cgacctccaa cacacaagca gggagcagat actggcttaa ctatgcggca
     1381 tcagagcaga ttgtactgag agtgcaccat aggggatcgg gagatctccc gatccgtcga
     1441 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1501 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1561 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1621 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1681 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1741 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1801 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1861 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1921 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1981 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2041 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2101 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     2161 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2221 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2281 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2341 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2401 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2461 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2521 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2581 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2641 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2701 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatacc gttcttctag
     2761 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2821 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2881 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2941 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3001 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3061 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3121 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3181 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3241 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3301 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3361 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     3421 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     3481 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     3541 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     3601 attacgccaa gctctagcta gaggtcgacg gtatacagac atgataagat acattgatga
     3661 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     3721 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttgggg tgggcgaaga
     3781 actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg aaaacgattc
     3841 cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg
     3901 gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact cgtcaagaag
     3961 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     4021 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     4081 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     4141 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     4201 catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     4261 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     4321 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     4381 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     4441 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     4501 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cctgcagttc
     4561 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     4621 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     4681 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     4741 cgatcctcat cctgtctctt gatcagatcc gaaaatggat atacaagctc ccgggagctt
     4801 tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc
     4861 agaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg
     4921 ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc
     4981 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     5041 acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     5101 tggggacttt ccacacccta actgacacac attccacaga attaattcgc gttaaatttt
     5161 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     5221 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     5281 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     5341 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     5401 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     5461 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     5521 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtggggatac
     5581 cccctagagc cccagctggt tctttccgcc tcagaagcca tagagcccac cgcatcccca
     5641 gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt cctgccccac cccacccccc
     5701 agaatagaat gacacctact cagacaatgc gatgcaattt cctcatttta ttaggaaagg
     5761 acagtgggag tggcaccttc cagggtcaag gaaggcacgg gggaggggca aacaacagat
     5821 ggctggcaac tagaaggcac agtcgaggct gatcagcggg tttaaacggg ccctctagac
     5881 tcgagttagg caggggccac ctgagaagtc tcggtcttag acacggtggt gctggcctca
     5941 tcgaattcac cggtgcgtcc accggtatcg atcagcagtg aatcattagt gctgcagttt
     6001 cgcccctggc tgtcaatatt gtcgcttggg actgttcctt ggtggccaac aaaatcctca
     6061 gtgccaggga ggtcctcgca caacagctta ttctcttttt cctgctccac gtggtatcct
     6121 gactgttcgc ccgtgttgcc attagaggag tatccgtttc cataagcctt caaggaagag
     6181 cgcctcagac acagcaattc ctggaatgcg attctgaagt ctgggctgcg gcagtaaatc
     6241 agagggttga acccagagtt cacgtagcca atccagttca ggaggatata cacctctttt
     6301 ctgatcaagt tgtcttgtat aacgtgaacg atattgacaa tgaaaaaggg gagccagcag
     6361 agagtgaatg tgcccataat tatccccaag gtcttcaggg ccttgtgttc cttcaagcaa
     6421 aatttggaag atctccgaag accatgtcca gttcttccgt cttgttcgac ctggctcaga
     6481 ttttgaacgt gaaatcggcc ttctgacttg tcgattttct ggagctgtct ttttgcctcc
     6541 tgaaaaaccc gagagtagac gaacaccatg ataaccagag gcacataaaa agagacaatt
     6601 gaactagcga tagcataggc ttgattagtg aagaagtcgc aacaggtttc gtttgcgtaa
     6661 caattgatag cttcttggtg tgtggcgcga taccagtgca tctgtatagg cagaaagctt
     6721 gtgagtcctg agacgatcca caccatgagt atgataaccc gagctttgtt cttagtcag
//
