LOCUS       sequence-430911-        6431 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 6431)
  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 6431)
  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: MC2R-DuET
FEATURES             Location/Qualifiers
     source          1..6431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     596..615
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(834..938)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1013..1032)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1204..1583
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1584..1787
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1738..1758
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1832..1851
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1832..1850
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2991..3008)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2997..3221
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3267..3695
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3354..3373)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3564..3585
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3704..3724)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3709..4038
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3889..4024
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3951..3970
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4105..4899
                     /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(4159..4178)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4769..4788
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5073..5194
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5110..5129)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5164..5183
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5243..5259)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5243..5259)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5256..5278)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5267..5283
                     /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(5291..5297,5298..5315,5316..5321))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5336..5357
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5474..5491)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5645..6233)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5725..5744)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6404..6431,1..764,765..833))
                     /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 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
       61 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
      121 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
      181 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
      241 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
      301 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
      361 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
      421 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
      481 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
      541 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
      601 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
      661 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
      721 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
      781 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
      841 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
      901 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
      961 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     1021 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     1081 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac atttgcatga
     1141 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     1201 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     1261 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     1321 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     1381 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     1441 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     1501 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     1561 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     1621 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     1681 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     1741 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     1801 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     1861 ctggctagcg tttaaactta agcttagcgg ccgcgccacc atgaagacga tcatcgccct
     1921 gagctacatc ttctgcctgg tattcgccga ctacaaggac gatgatgacg ccagcatcga
     1981 tatgaagcat atcatcaatt cctatgagaa tatcaataac acggccagga acaatagcga
     2041 ctgtcccagg gtagttctgc ctgaagagat attcttcacc atatccatag tgggcgtact
     2101 ggaaaacctc atcgttttgc tggcggtctt taagaataag aacctgcagg ctccaatgta
     2161 cttttttatt tgctccctgg ccattagcga tatgctgggg agcctctata agatcctgga
     2221 gaacatcctt ataattttgc ggaacatggg ttacctcaaa cctcgcggat ctttcgaaac
     2281 caccgccgat gatatcatag actccctgtt tgtcctgtcc ttgctgggct caattttcag
     2341 cctgagcgtt attgccgccg accgctacat tacaatcttc cacgcccttc gctatcactc
     2401 catcgtgact atgagacgga cagttgtggt actcacggtc atttggactt tttgtaccgg
     2461 cactggaatc accatggtca tcttcagtca ccacgtccca acggttatta cgttcacatc
     2521 cctgttccct ctgatgctgg tcttcattct ctgtctgtac gtacacatgt tcctgctcgc
     2581 ccggtcccat accaggaaga tatccacgtt gccccgagct aacatgaagg gagccatcac
     2641 ccttaccatt ctgctgggtg tcttcatctt ttgctgggct ccctttgtgc tccacgtgct
     2701 gttgatgacc ttctgtccgt caaatccata ctgtgcttgt tatatgtcac tgttccaggt
     2761 gaatggaatg cttatcatgt gtaacgcagt gatagatcca tttatatacg cttttcgaag
     2821 ccccgaactg agagatgctt ttaagaaaat gatattctgc agtaggtatt ggatcgatac
     2881 cggtggacgc accggtgaat tcgatgaggc cagcaccacc gtgtctaaga ccgagacttc
     2941 tcaggtggcc cctgcctaat ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     3001 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3061 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3121 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3181 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3241 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3301 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3361 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3421 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3481 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3541 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3601 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3661 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3721 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3781 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3841 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3901 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3961 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4021 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     4081 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     4141 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4201 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4261 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4321 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4381 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4441 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4501 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4561 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4621 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4681 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4741 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4801 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4861 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4921 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4981 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5041 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     5101 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5161 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     5221 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5281 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5341 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5401 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5461 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5521 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5581 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5641 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5701 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5761 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5821 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5881 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5941 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6001 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6061 ggcctaacta cggctacact agaagaacgg tatttggtat ctgcgctctg ctgaagccag
     6121 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6181 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6241 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6301 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6361 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6421 gtgaggcacc t
//
