LOCUS       sequence-430820-        6623 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 6623)
  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 6623)
  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: GPR183-DuET
FEATURES             Location/Qualifiers
     source          1..6623
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     554..573
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(792..896)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(971..990)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1162..1541
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1542..1745
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1696..1716
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1790..1809
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1790..1808
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3141..3158)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3147..3371
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3417..3845
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3504..3523)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3714..3735
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3854..3874)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3859..4188
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4039..4174
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4101..4120
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4255..5049
                     /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(4309..4328)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4919..4938
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5223..5344
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5260..5279)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5314..5333
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5393..5409)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5393..5409)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5406..5428)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5417..5433
                     /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(5441..5447,5448..5465,5466..5471))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5486..5507
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5624..5641)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5795..6383)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5875..5894)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6554..6623,1..722,723..791))
                     /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"
ORIGIN
        1 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
       61 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      121 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      181 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      241 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      301 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      361 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      421 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      481 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      541 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      601 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      661 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      721 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
      781 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
      841 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
      901 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
      961 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     1021 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     1081 tgaccgacat ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     1141 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     1201 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1261 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1321 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1381 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1441 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1501 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1561 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1621 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1681 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1741 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     1801 actataggga gacccaagct ggctagcgtt taaacttaag cttagcgcca ccatgaagac
     1861 gatcatcgcc ctgagctaca tcttctgcct ggtattcgcc gactacaagg acgatgatga
     1921 cgccagcatc gatatggaca tccagatggc taacaatttc acgcctccct ctgcgacccc
     1981 tcaggggaat gattgtgatc tttacgccca ccactctacg gcccgaattg ttatgcctct
     2041 tcactatagc ctggttttca tcattgggct ggtaggtaac ttgctggccc ttgtggtgat
     2101 cgtgcaaaac agaaaaaaaa tcaattccac cactttgtac agcaccaacc tggtcattag
     2161 cgacattctg tttaccacag ccctgccgac acgaatcgct tattacgcca tgggattcga
     2221 ctggcggatc ggtgatgcct tgtgccggat tacagccctc gtcttttata tcaacacata
     2281 tgccggagtg aacttcatga catgcctgag catcgaccgc tttatagctg ttgttcaccc
     2341 tctccgctat aacaagataa aacgcatcga gcatgcgaag ggtgtatgca tcttcgtgtg
     2401 gatattggtc ttcgcccaga ccttgcctct cttgattaac ccaatgagca aacaagaggc
     2461 cgagaggatc acatgcatgg agtacccaaa ttttgaggag actaagtctc ttccttggat
     2521 actgctgggg gcctgtttca ttgggtacgt gctgccactg attatcattc tcatttgcta
     2581 ctcccagatc tgctgcaaac ttttccgaac agccaagcaa aaccccctta ccgaaaaaag
     2641 cggcgtcaac aagaaagcac tgaatactat catcctcatt atcgttgtct ttgtcctgtg
     2701 cttcactccc tatcatgtcg ccatcattca gcacatgata aagaagctgc gcttctccaa
     2761 tttcttggag tgtagtcagc ggcatagctt tcagatctcc ctccatttca ctgtttgcct
     2821 gatgaatttc aattgttgca tggacccttt tatatacttt tttgcctgta aagggtacaa
     2881 aaggaaagtc atgcgcatgc tgaagcggca agtgagcgtt tctatcagtt ctgctgtgaa
     2941 atcagccccg gaagaaaatt ctagggagat gacagaaaca cagatgatga tccacagcaa
     3001 atccagtaat gggaagatcg ataccggtgg acgcaccggt gaattcgatg aggccagcac
     3061 caccgtgtct aagaccgaga cttctcaggt ggcccctgcc taactcgagt ctagagggcc
     3121 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3181 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3241 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3301 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3361 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3421 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3481 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3541 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3601 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3661 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3721 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3781 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3841 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3901 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3961 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4021 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4081 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     4141 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     4201 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4261 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4321 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4381 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     4441 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4501 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4561 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4621 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     4681 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     4741 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     4801 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4861 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4921 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4981 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     5041 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     5101 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5161 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5221 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5281 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5341 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5401 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5461 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5521 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5581 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5641 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5701 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5761 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5821 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5881 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5941 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6001 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6061 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6121 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6181 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacgg
     6241 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6301 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6361 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6421 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6481 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6541 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6601 ttcgttcatc catagttgcc tga
//
