LOCUS       sequence-430817-        6659 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 6659)
  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 6659)
  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: GPR173-DuET
FEATURES             Location/Qualifiers
     source          1..6659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      245..833
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     734..753
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     987..1004
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1121..1142
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1157..1162,1163..1180,1181..1187)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1195..1211
                     /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     1200..1222
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1219..1235
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1219..1235
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1284..1405
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1295..1314)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1349..1368
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1579..2373)
                     /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(1690..1709)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2300..2319
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2440..2769)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2454..2589
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2508..2527)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2754..2774
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2783..3211)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2893..2914)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3105..3124
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3257..3481
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3470..3487
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4855..4874)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4856..4874)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4919..5122)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4948..4968)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5123..5502
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5674..5693
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5768..5872
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5873..5941,5942..6659,1..74)
                     /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(6091..6110)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
       61 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
      121 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
      181 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
      241 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
      301 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
      361 gcttcagcag agcgcagata ccaaataccg ttcttctagt gtagccgtag ttaggccacc
      421 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
      481 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
      541 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
      601 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
      661 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
      721 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
      781 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
      841 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
      901 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
      961 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     1021 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     1081 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     1141 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     1201 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctctagctag
     1261 aggtcgacgg tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag
     1321 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     1381 cattataagc tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc
     1441 gctggaggat catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga
     1501 aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt
     1561 cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg
     1621 cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag
     1681 ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag
     1741 ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca
     1801 ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc
     1861 gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag
     1921 accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg
     1981 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt
     2041 ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag
     2101 ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt
     2161 ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc
     2221 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga
     2281 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg
     2341 agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg
     2401 atcagatccg aaaatggata tacaagctcc cgggagcttt ttgcaaaagc ctaggcctcc
     2461 aaaaaagcct cctcactact tctggaatag ctcagaggca gaggcggcct cggcctctgc
     2521 ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag
     2581 gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga gatgcatgct
     2641 ttgcatactt ctgcctgctg gggagcctgg ggactttcca cacctggttg ctgactaatt
     2701 gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc cacaccctaa
     2761 ctgacacaca ttccacagaa ttaattcgcg ttaaattttt gttaaatcag ctcatttttt
     2821 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     2881 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     2941 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3001 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3061 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3121 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3181 gccgcgctta atgcgccgct acagggcgcg tggggatacc ccctagagcc ccagctggtt
     3241 ctttccgcct cagaagccat agagcccacc gcatccccag catgcctgct attgtcttcc
     3301 caatcctccc ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc
     3361 agacaatgcg atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc
     3421 agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca
     3481 gtcgaggctg atcagcgggt ttaaacgggc cctctagact cgagttaggc aggggccacc
     3541 tgagaagtct cggtcttaga cacggtggtg ctggcctcat cgaattcacc ggtgcgtcca
     3601 ccggtatcga tcataacgca ataaggttct ctcggggcag gtgctccgcc agtcccccaa
     3661 cagggggcat gtgtgcgcag acattttttc aaatccttat tcaggagaaa gcagacaatt
     3721 ggattcactg cagcctgggc gaaggacatc cacacagcgg tggccagata cctgtgtggc
     3781 acggcacaag ctttgacgaa gacgcgccag tagcatgcca caatgtaggg gctccacaga
     3841 agcaggaaga gcagagtaat cgcatagaac attcgcccaa gctgcttctc acccttaact
     3901 tcgtccatac ccagcaacct ccttgaagcg gcgtggccgt tctggcggat gccaaggagt
     3961 gtaggtggca taggacctcg tccgaaccca gcgatccaat tggcagcagc ctggccagtg
     4021 gccccaggtc catggaaggt ccaattctga cttatggctg gtaccatttg gacaggcttc
     4081 atttttctat ggcgatattc gaagaggagc aacttgccat aaacagcatg tgtagcggcc
     4141 atcaaaactg ccaacataag catgaatccc agtgtatcgt tagccttgaa gtaccgatgc
     4201 tcaaagatac actgatcctc ctctctaata aatttatagg tgcctacatc gaagacagga
     4261 ggaaaggcca tggccacgct cagggtccac gccatgcaaa tcacggccgc acaagtccac
     4321 agcgtcatcc tcttagcgta aaatctgtga tgggcaatag ccatatagcg tgtcacagaa
     4381 atacagaaga gcatgaaggc ggcatggaag cagaaaagga cggccataaa cgccacgatt
     4441 ttgcatgaca gtgcgctgaa ggtccagctt gagccatggc gtacagaagc gaggacgaaa
     4501 ggaaagcata cagcgctccg gatcccgtct gccaaacaga ggtccaaaag gaaataataa
     4561 ggggctttat ggagagctct ctctttcaac accagcaggc tcaggatagc gtttccagcc
     4621 agagagacgc acataatcag tccaagcagg accagtttca cgtatgcact ggcgctgggg
     4681 ggggacagtg cgccgctcac ttcttcaggt tcgcctgtgg tgtttgccat atcgatgctg
     4741 gcgtcatcat cgtccttgta gtcggcgaat accaggcaga agatgtagct cagggcgatg
     4801 atcgtcttca tggtggcgct aagcttaagt ttaaacgcta gccagcttgg gtctccctat
     4861 agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt agccagagag
     4921 ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc aatggggcgg
     4981 agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa ctcccattga
     5041 cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga
     5101 tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag atgtactgcc
     5161 aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc catttaccgt
     5221 cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg
     5281 cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta
     5341 tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg
     5401 ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga actaatgacc
     5461 ccgtaattga ttactattaa taactagtca ataatcaatg tcaacgcgta tatctggccc
     5521 gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg caaatgtcgg
     5581 tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg acctccaaca
     5641 cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt gtactgagag
     5701 tgcaccatag gggatcggga gatctcccga tccgtcgacg tcaggtggca cttttcgggg
     5761 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5821 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5881 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5941 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     6001 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     6061 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     6121 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     6181 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6241 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6301 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6361 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6421 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6481 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6541 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6601 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacgg
//
