LOCUS       sequence-430798-        6593 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 6593)
  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 6593)
  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: FPR2-DuET
FEATURES             Location/Qualifiers
     source          1..6593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..23
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(1325..1344)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1326..1344)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1389..1592)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1418..1438)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1593..1972
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2144..2163
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2238..2342
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2343..2411,2412..3203)
                     /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(2561..2580)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3374..3962
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3863..3882
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4116..4133
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4250..4271
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4286..4291,4292..4309,4310..4316)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4324..4340
                     /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     4329..4351
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4348..4364
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4348..4364
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4413..4534
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4424..4443)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4478..4497
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4708..5502)
                     /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(4819..4838)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5429..5448
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5569..5898)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5583..5718
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5637..5656)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5883..5903
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5912..6340)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6022..6043)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6234..6253
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    join(6386..6593,1..17)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gcaactagaa ggcacagtcg aggctgatca gcgggtttaa acgggccctc tagactcgag
       61 ttaggcaggg gccacctgag aagtctcggt cttagacacg gtggtgctgg cctcatcgaa
      121 ttcaccggtg cgtccaccgg tatcgatcat cgcttggagt tctgtctcag caggaggaga
      181 agcggagttg gcggcggtgt cgttcgtggg cgcactatct tcgctcaggg ctctctccag
      241 tgaggtagga aggctgtgaa tcagccgttc tcggaagtcc tggcccacaa aaacatacag
      301 cattgggttc aggcaagagt taaaaaaagc cagactgctg gtagggttta cgagaatgtc
      361 aattatttta tacttcccat agaacaacat ttctttcagc cacacagttc ccagcagtgc
      421 caccagctgg aaagggaacc agcatataaa gaaacttgca actacggctg tcaatacccg
      481 gagaggcctg gaagacttga tcataccttt cttatggatc ttagccgcga tgaggccgta
      541 gcagatagcc actatagaca tcggcaggga gaagccaatc acgaatctga ttattcctct
      601 ggctgtcaac atggttatcg caaccttcaa tcgctcctca ggcgtacccc cccagcttgc
      661 gaaattaaat gtgcagtatg tgtcgccatt tgggatggtc acggtggtga gaaacaagaa
      721 aactggcagg gtaagcacca gggccaagat ccatggacca actatcactt tcatagcaag
      781 gctgacggtt ctgtgattct gggcccagac tgggtgcagt acacaaatac acctatcgag
      841 tgcaataaag cctatcagga agacactacc aaaaagattg atgtccacca caatgtggat
      901 gagcttgcac aggaaccacc cgaaaggcca tttctcgccc atggccatag acacaatgag
      961 aaacgggaga gtagcagtaa aggaaaaatc ggccagggcc aggttgaggt agcaaatggt
     1021 agtcaccgtg cgagtcattc tgaaccctgc cacccagatt accaacccgt taccaagaac
     1081 accgagcaca aaagtcacgc caagtacgac aagcgggagg attcgcagga ctgtgtagcc
     1141 cgctgattcg taagacacct cttcgtactc attcagggga gtagagaaat tggtttccat
     1201 atcgatgctg gcgtcatcat cgtccttgta gtcggcgaat accaggcaga agatgtagct
     1261 cagggcgatg atcgtcttca tggtggcgct aagcttaagt ttaaacgcta gccagcttgg
     1321 gtctccctat agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt
     1381 agccagagag ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc
     1441 aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa
     1501 ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca
     1561 cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag
     1621 atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc
     1681 catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact
     1741 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt
     1801 ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt
     1861 cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga
     1921 actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg tcaacgcgta
     1981 tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg
     2041 caaatgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg
     2101 acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt
     2161 gtactgagag tgcaccatag gggatcggga gatctcccga tccgtcgacg tcaggtggca
     2221 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2281 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2341 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     2401 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     2461 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     2521 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     2581 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     2641 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     2701 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     2761 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     2821 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     2881 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     2941 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3001 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     3061 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3121 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     3181 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3241 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3301 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3361 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3421 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3481 aggtaactgg cttcagcaga gcgcagatac caaataccgt tcttctagtg tagccgtagt
     3541 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3601 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3661 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3721 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3781 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3841 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3901 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3961 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4021 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4081 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4141 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     4201 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     4261 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     4321 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     4381 tctagctaga ggtcgacggt atacagacat gataagatac attgatgagt ttggacaaac
     4441 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     4501 atttgtaacc attataagct gcaataaaca agttggggtg ggcgaagaac tccagcatga
     4561 gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg aagcccaacc
     4621 tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt
     4681 cggtcatttc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc
     4741 gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc
     4801 gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
     4861 cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt
     4921 cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt
     4981 gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg
     5041 atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg
     5101 gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat
     5161 ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc
     5221 caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac
     5281 gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc
     5341 ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc
     5401 ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca
     5461 agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc
     5521 tgtctcttga tcagatccga aaatggatat acaagctccc gggagctttt tgcaaaagcc
     5581 taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggcag aggcggcctc
     5641 ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg
     5701 cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag
     5761 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc
     5821 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     5881 acaccctaac tgacacacat tccacagaat taattcgcgt taaatttttg ttaaatcagc
     5941 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     6001 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     6061 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     6121 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     6181 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     6241 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     6301 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ggggataccc cctagagccc
     6361 cagctggttc tttccgcctc agaagccata gagcccaccg catccccagc atgcctgcta
     6421 ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga
     6481 cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg
     6541 gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctg
//
