LOCUS       sequence-431817-        6759 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 6759)
  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 6759)
  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: HTR4-DuET
FEATURES             Location/Qualifiers
     source          1..6759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      101..689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     590..609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     843..860
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    977..998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1013..1018,1019..1036,1037..1043)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1051..1067
                     /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     1056..1078
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1075..1091
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1075..1091
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1140..1261
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1151..1170)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1205..1224
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1435..2229)
                     /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(1546..1565)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2156..2175
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2296..2625)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2310..2445
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2364..2383)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2610..2630
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2639..3067)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2749..2770)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2961..2980
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3113..3337
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3326..3343
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4811..4830)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4812..4830)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4875..5078)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4904..4924)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5079..5458
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5630..5649
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5724..5828
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5829..5897,5898..6689)
                     /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(6047..6066)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
       61 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
      121 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
      181 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
      241 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
      301 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
      361 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
      421 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
      481 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
      541 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
      601 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
      661 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
      721 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
      781 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
      841 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
      901 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
      961 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     1021 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     1081 acagctatga ccatgattac gccaagctct agctagaggt cgacggtata cagacatgat
     1141 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     1201 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     1261 tggggtgggc gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt
     1321 cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc
     1381 gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa ccccagagtc ccgctcagaa
     1441 gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta
     1501 aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc
     1561 caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga
     1621 aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag
     1681 atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc
     1741 ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc
     1801 tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg
     1861 cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga
     1921 caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac
     1981 aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc
     2041 ctcgtcctgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg
     2101 cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca
     2161 gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg
     2221 ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gatccgaaaa tggatataca
     2281 agctcccggg agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg
     2341 gaatagctca gaggcagagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc
     2401 atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg
     2461 cgggactatg gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga
     2521 gcctggggac tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct
     2581 gcctgctggg gagcctgggg actttccaca ccctaactga cacacattcc acagaattaa
     2641 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     2701 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     2761 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     2821 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     2881 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     2941 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     3001 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3061 ggcgcgtggg gataccccct agagccccag ctggttcttt ccgcctcaga agccatagag
     3121 cccaccgcat ccccagcatg cctgctattg tcttcccaat cctccccctt gctgtcctgc
     3181 cccaccccac cccccagaat agaatgacac ctactcagac aatgcgatgc aatttcctca
     3241 ttttattagg aaaggacagt gggagtggca ccttccaggg tcaaggaagg cacgggggag
     3301 gggcaaacaa cagatggctg gcaactagaa ggcacagtcg aggctgatca gcgggtttaa
     3361 acgggccctc tagactcgag cggccgccac tgtgctggat atctgcagaa ttccaccaca
     3421 ctggactagt ggatccgagc tcggtaccaa gcttttaggc aggggccacc tgagaagtct
     3481 cggtcttaga cacggtggtg ctggcctcat cgaattcacc ggtgcgtcca ccggtatcga
     3541 tagtatctga aggttgagcc gcgacgagtg gagaggtagc gggaggatga cactgggact
     3601 cccattgacc gccgcactcg acggcgtcgc gcagaacatg tgtgctaccg ttgatagttg
     3661 tagttgagca agggacggtc tgtcccagga tagaaggtct cctataccgt tcatcatcgc
     3721 aacacaatat aataagaaat gcgcgcctga aggatttatt aaggaaggca tagagaaaag
     3781 gattcaggcc gctattgata tagcccaacc agaggaaggc ggtccacacc tgtccgggca
     3841 ctgtgtagtc aatgaaagga tcgacgatgt tggttacaaa aaatggtgcc cagcacaagc
     3901 agaagcatcc cataataatg caaagagttt ttgctgcttt ggtctcagtg cgcatcctat
     3961 gggttgaatg ctgatctgct gactgaggcc tactttcaga actggctccg gctctctgaa
     4021 gcatctgtat ctggtgtgcg tgttcctttg ctgtcacgta gattctataa taggccagca
     4081 ccataaggag aaagggaatg tagaatgcga ccacgctgca agttatagcg tacggtttgt
     4141 ttaccatgaa tacacagtag gtagagttgg aattttgatt gaacttgcgc ttctcaatga
     4201 ggtctattat tccgatatta ttccaaccct gcataatcgg caagaaactt ataaaggtgg
     4261 ggataaccca gcaacccccg agcatcaggg cgatcctcaa aggtgtcatt ttattcctgt
     4321 acaccagtgg ctggcagcag attgcgtaat atctgtccag gctaatacag caaaggtgaa
     4381 aaatgctggc agtagtcagc agaacatcca atgaagttct caccaggcag aacacttccc
     4441 catagatcca aatgtcctgc accagttcga tggctccgaa tggcatcacc agcacactaa
     4501 ccaggaggtc tgcaaaggcg agagacacta tgaaatagtt agtcttgatt ttccgcagct
     4561 gcctatccca acaaacggct accatgacga gcaggttacc caagattgcc atcaggatca
     4621 cagtggacag gaaggtgagc agcaccacct tctcaacact gccaaatcct tcttcactgc
     4681 taacgttagc atccagctta tccatatcga tgctggcgtc atcatcgtcc ttgtagtcgg
     4741 cgaataccag gcagaagatg tagctcaggg cgatgatcgt cttcatggtg gcgctagcca
     4801 gcttgggtct ccctatagtg agtcgtatta atttcgataa gccagtaagc agtgggttct
     4861 ctagttagcc agagagctct gcttatatag acctcccacc gtacacgcct accgcccatt
     4921 tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa
     4981 aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc
     5041 tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat
     5101 acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag
     5161 gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga
     5221 tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc
     5281 cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg
     5341 ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg
     5401 ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa tcaatgtcaa
     5461 cgcgtatatc tggcccgtac atcgcgaagc agcgcaaaac gcctaaccct aagcagattc
     5521 ttcatgcaat tgtcggtcaa gccttgcctt gttgtagctt aaattttgct cgcgcactac
     5581 tcagcgacct ccaacacaca agcagggagc agatactggc ttaactatgc ggcatcagag
     5641 cagattgtac tgagagtgca ccatagggga tcgggagatc tcccgatccg tcgacgtcag
     5701 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5761 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5821 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5881 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5941 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     6001 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     6061 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     6121 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     6181 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     6241 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     6301 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6361 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6421 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6481 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6541 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6601 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6661 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6721 agattgattt aaaacttcat ttttaattta aaaggatct
//
