LOCUS       sequence-430797-        6590 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 6590)
  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 6590)
  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: FPR1-DuET
FEATURES             Location/Qualifiers
     source          1..6590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      98..686
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     587..606
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     840..857
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    974..995
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1010..1015,1016..1033,1034..1040)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1048..1064
                     /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     1053..1075
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1072..1088
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1072..1088
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1137..1258
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1148..1167)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1202..1221
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1432..2226)
                     /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(1543..1562)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2153..2172
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2293..2622)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2307..2442
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2361..2380)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2607..2627
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2636..3064)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2746..2767)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2958..2977
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3110..3334
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3323..3340
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4639..4658)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4640..4658)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4703..4906)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4732..4752)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4907..5286
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5458..5477
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5552..5656
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5657..5725,5726..6517)
                     /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(5875..5894)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
       61 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
      121 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
      181 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
      241 ccgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
      301 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
      361 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
      421 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
      481 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
      541 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
      601 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
      661 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
      721 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
      781 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
      841 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
      901 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
      961 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     1021 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     1081 gctatgacca tgattacgcc aagctctagc tagaggtcga cggtatacag acatgataag
     1141 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     1201 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg
     1261 ggtgggcgaa gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc
     1321 ggaaaacgat tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgtg
     1381 atggcaggtt gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg ctcagaagaa
     1441 ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag
     1501 cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa
     1561 cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa
     1621 gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc
     1681 ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg
     1741 atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg
     1801 ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag
     1861 ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag
     1921 gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac
     1981 gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc
     2041 gtcctgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc
     2101 ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc
     2161 atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc
     2221 aatcatgcga aacgatcctc atcctgtctc ttgatcagat ccgaaaatgg atatacaagc
     2281 tcccgggagc tttttgcaaa agcctaggcc tccaaaaaag cctcctcact acttctggaa
     2341 tagctcagag gcagaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg
     2401 gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg
     2461 gactatggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     2521 tggggacttt ccacacctgg ttgctgacta attgagatgc atgctttgca tacttctgcc
     2581 tgctggggag cctggggact ttccacaccc taactgacac acattccaca gaattaattc
     2641 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     2701 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     2761 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     2821 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     2881 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     2941 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     3001 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     3061 gcgtggggat accccctaga gccccagctg gttctttccg cctcagaagc catagagccc
     3121 accgcatccc cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc
     3181 accccacccc ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt
     3241 tattaggaaa ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg
     3301 caaacaacag atggctggca actagaaggc acagtcgagg ctgatcagcg ggtttaaacg
     3361 ggccctctag actcgagtta ggcaggggcc acctgagaag tctcggtctt agacacggtg
     3421 gtgctggcct catcgaattc accggtgcgt ccaccggtat cgatcttagc ctggagctca
     3481 acttccgcac tgggcagggt actgttagta gcagtgtcac tcgtctgcgt agagtcttcg
     3541 gtcagagccc tttccaggga ggctggcagt gcgtgaatga gtcgttcccg aaagtcctgt
     3601 cccataaaca catacagcat tggattaaga caagagttaa aaaatgccag agcacttgtt
     3661 acatccacgg caatgccgat ttccttatac ataccctgca gcagttccct tatccgcaca
     3721 gttgcaataa gggccacgac ctgataaggg ctccagcaca ggaaaaatgc ggcagcaacg
     3781 aaactcagca cccggagagg tctgctggac ttgatcagtc cctgtttgtg tatcttcgtg
     3841 gctatgaggc cgtagctcac ggccactata gacattgggg cggagaaacc aatgatgaat
     3901 cttattatcc cgcgcacggt caacatggcc acggcgacat tgattctttc tttggggtca
     3961 tttgtccagg gagagaaatt aaatgtgcaa gcgacggtgc cggtctttcc cgggactgtg
     4021 gtgactcgga taataacagg gagggtcagc agcagcgcca tcacccatgg gccaatgatg
     4081 actttcttgg ccagggacac ggttcggtga ttctgagtcc agacgggatg aaggacacac
     4141 acgcagcggt ccagggctat cagagcgatg aggaacacgc taccgaacag gttaatgtcg
     4201 acaatggtga aaacaaactt acacagaaac cagccgaaag gccaatgtcc gcccatggcc
     4261 tttcgcacca taaaaaaggg aagggtagaa gtaaagcaga aatctgccac agccaggttc
     4321 agataggata tggtggtcac ggtgtgggtc atcctaaagc ctgcgaccca gatcacaagc
     4381 ccattcccaa gtactccgag cacaaatgtc actgcgaaga ccaagtaagt gataatgtcc
     4441 aagaacagat agcctgcgct gacggccggc gtcccccctg aaatatttgt aggcaggctg
     4501 ctattggtct ccatatcgat gctggcgtca tcatcgtcct tgtagtcggc gaataccagg
     4561 cagaagatgt agctcagggc gatgatcgtc ttcatggtgg cgctaagctt aagtttaaac
     4621 gctagccagc ttgggtctcc ctatagtgag tcgtattaat ttcgataagc cagtaagcag
     4681 tgggttctct agttagccag agagctctgc ttatatagac ctcccaccgt acacgcctac
     4741 cgcccatttg cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt
     4801 ggtgccaaaa caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt
     4861 caaaccgcta tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga
     4921 tgactaatac gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat
     4981 aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat
     5041 acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat
     5101 ggaaagtccc tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg
     5161 ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca
     5221 tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc
     5281 aatgtcaacg cgtatatctg gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa
     5341 gcagattctt catgcaaatg tcggtcaagc cttgccttgt tgtagcttaa attttgctcg
     5401 cgcactactc agcgacctcc aacacacaag cagggagcag atactggctt aactatgcgg
     5461 catcagagca gattgtactg agagtgcacc ataggggatc gggagatctc ccgatccgtc
     5521 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5581 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     5641 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5701 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5761 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5821 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     5881 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5941 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6001 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6061 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6121 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6181 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6241 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6301 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6361 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6421 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6481 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6541 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
//
