LOCUS       sequence-430851-        6617 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 6617)
  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 6617)
  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: HRH2-DuET
FEATURES             Location/Qualifiers
     source          1..6617
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(400..417)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    406..630
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      676..1104
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(763..782)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     973..994
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1113..1133)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1118..1447
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1298..1433
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1360..1379
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1514..2308
                     /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(1568..1587)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2178..2197
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2482..2603
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2519..2538)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2573..2592
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2652..2668)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2652..2668)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2665..2687)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2676..2692
                     /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(2700..2706,2707..2724,2725..2730))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2745..2766
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2883..2900)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3054..3642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3134..3153)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3813..4604,4605..4673))
                     /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     4436..4455
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4674..4778)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4853..4872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5044..5423
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5424..5627
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5578..5598
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5672..5691
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5672..5690
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tcctggaggc catcgtcctg tggctgggct acgccaacag tgctcttaac ccaatcctct
       61 atgcggccct caaccgggat tttaggacag ggtaccagca gcttttttgc tgcaggcttg
      121 caaatcggaa ttcccataag acatccctcc ggagtaacgc cagtcagctc agccgaacac
      181 agagcaggga gccgcggcag caggaggaaa aacctcttaa gctccaggtt tggtcaggaa
      241 cagaggtgac tgcccctcaa ggtgctaccg acagaatcga taccggtgga cgcaccggtg
      301 aattcgatga ggccagcacc accgtgtcta agaccgagac ttctcaggtg gcccctgcct
      361 aactcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
      421 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
      481 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
      541 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
      601 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
      661 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      721 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      781 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      841 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
      901 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
      961 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1021 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1081 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     1141 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     1201 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     1261 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     1321 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     1381 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     1441 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     1501 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     1561 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     1621 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     1681 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     1741 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     1801 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     1861 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     1921 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     1981 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     2041 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     2101 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     2161 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     2221 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     2281 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     2341 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     2401 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     2461 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     2521 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2581 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     2641 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2701 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2761 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2821 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2881 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2941 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3001 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3061 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3121 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3181 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3241 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3301 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3361 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3421 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3481 ggctacacta gaagaacggt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3541 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3601 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3661 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3721 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3781 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3841 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3901 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3961 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4021 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4081 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     4141 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4201 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4261 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     4321 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4381 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4441 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4501 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4561 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4621 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4681 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4741 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4801 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     4861 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     4921 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac atttgcatga
     4981 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     5041 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     5101 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     5161 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     5221 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     5281 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     5341 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     5401 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     5461 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     5521 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     5581 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     5641 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     5701 ctggctagcg tttaaactta agcttagcgc caccatgaag acgatcatcg ccctgagcta
     5761 catcttctgc ctggtattcg ccgactacaa ggacgatgat gacgccagca tcgatatggc
     5821 tcctaatggg actgcctcct ccttctgcct tgactctact gcctgcaaaa taactattac
     5881 agtggtactc gccgtcctga tactgattac tgttgcgggt aatgtcgtag tgtgtctggc
     5941 cgtcggcctt aaccggagac tgagaaacct tactaactgc ttcattgtga gcctggcgat
     6001 cactgatctc ctcctgggcc tcctggtgtt gccctttagt gcaatttacc agctgtcatg
     6061 taagtggtcc ttcggaaaag tattctgcaa tatttatacg agcctggatg tgatgctgtg
     6121 cacggcctcc atactgaatc tgttcatgat ttcccttgat cggtattgcg ccgtgatgga
     6181 tcccctgcgg tacccagtct tggtgacccc cgttagagtg gcgattagtc tggtcctcat
     6241 ttgggttatc agcattaccc tctccttcct gagcatccat ctgggctgga actccagaaa
     6301 cgagacctct aaggggaacc acacaaccag taagtgcaag gtgcaggtta acgaggttta
     6361 cgggctggta gatggccttg tgacttttta cttgcccctc ctgattatgt gtataactta
     6421 ttaccggatt ttcaaggtgg ccagagacca ggccaaacgc ataaaccaca tcagctcctg
     6481 gaaagccgca accatccgcg agcacaaagc aacggtgact ctcgctgctg tgatgggtgc
     6541 cttcatcatc tgctggttcc cttactttac cgcctttgtg taccggggcc tgcgaggcga
     6601 cgacgctatt aacgagg
//
