LOCUS       sequence-430801-        6971 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 6971)
  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 6971)
  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: GCGR-DuET
FEATURES             Location/Qualifiers
     source          1..6971
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     508..527
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(746..850)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(925..944)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1116..1495
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1496..1699
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1650..1670
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1744..1763
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1744..1762
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3443..3460)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3449..3673
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3719..4147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3806..3825)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4016..4037
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4156..4176)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4161..4490
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4341..4476
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4403..4422
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4557..5351
                     /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(4611..4630)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5221..5240
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5525..5646
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5562..5581)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5616..5635
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5695..5711)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5695..5711)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5708..5730)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5719..5735
                     /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(5743..5749,5750..5767,5768..5773))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5788..5809
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5926..5943)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6097..6685)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6177..6196)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6856..6971,1..676,677..745))
                     /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 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
       61 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
      121 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
      181 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
      241 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
      301 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
      361 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
      421 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
      481 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
      541 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
      601 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
      661 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
      721 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
      781 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
      841 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
      901 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
      961 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     1021 aacaaggcaa ggcttgaccg acatttgcat gaagaatctg cttagggtta ggcgttttgc
     1081 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
     1141 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     1201 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     1261 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     1321 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     1381 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     1441 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     1501 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     1561 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     1621 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     1681 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
     1741 aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttagc
     1801 gccaccatga agacgatcat cgccctgagc tacatcttct gcctggtatt cgccgactac
     1861 aaggacgatg atgacgccag catcgatatg cctccctgcc aaccacagag gcccttgctt
     1921 ctcctgctcc ttctgctcgc atgtcagccc caggtgccct ctgcccaggt gatggatttt
     1981 ctgttcgaaa agtggaaact gtacggcgat cagtgccacc ataacctgtc actcctgccg
     2041 ccacccaccg aactcgtatg caatcgcact ttcgacaaat attcctgctg gcccgatacc
     2101 cccgccaata ctaccgccaa tatatcatgt ccatggtatc tgccttggca ccacaaggtg
     2161 caacacaggt ttgtgttcaa aaggtgcggc cctgacggac agtgggtcag gggaccacga
     2221 ggccagccct ggcgggatgc aagccagtgc caaatggatg gagaggaaat agaggtgcaa
     2281 aaggaagtgg ctaagatgta tagcagcttc caggtgatgt acacagtggg gtattctctg
     2341 tctttgggcg cgctcctcct ggctctggca attctgggtg gcctgagtaa attgcattgc
     2401 acacggaatg cgatccacgc aaatcttttc gcttcctttg tcctgaaagc gtctagtgtt
     2461 ctggttatcg acggcctgct gaggacgagg tactctcaga agataggtga tgatcttagc
     2521 gtgtcaactt ggttgagcga cggtgccgtt gccgggtgtc gagtggcggc agtctttatg
     2581 caatacggca ttgtcgccaa ctactgctgg ttgctcgtcg aaggccttta cctccacaac
     2641 ttgctggggc tggccactct gcccgagaga tcctttttct cactttacct gggcatcgga
     2701 tggggagccc caatgctgtt cgtggtgcca tgggccgttg tcaaatgtct gtttgagaac
     2761 gtgcagtgct ggacctccaa tgacaatatg ggcttttggt ggattttgcg attccctgtg
     2821 ttcctggcta tccttatcaa ctttttcatc tttgtgagaa tcgtacagct tttggtcgca
     2881 aagctgaggg ctaggcaaat gcaccatacc gattataagt tcaggctggc caaaagcacc
     2941 ctcactctga ttcccctgct cggcgtccac gaagttgtct ttgctttcgt gactgatgaa
     3001 cacgcacagg gaaccttgcg cagtgcaaag cttttctttg acctgttcct gagtagcttc
     3061 caaggactgt tggtcgcggt gctgtactgt ttcctgaata aggaagtgca gtccgagctg
     3121 agacgaagat ggcatcggtg gagactcgga aaggtccttt gggaagagag gaacacctct
     3181 aaccaccggg ccagctcctc tcccggtcac ggaccaccgt ccaaggaact gcaatttggg
     3241 cgcggaggcg gaagccagga ctcttcagcg gagacgccgt tggctggcgg tctccctcgg
     3301 ctcgccgagt cacctttcat cgataccggt ggacgcaccg gtgaattcga tgaggccagc
     3361 accaccgtgt ctaagaccga gacttctcag gtggcccctg cctaactcga gtctagaggg
     3421 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     3481 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     3541 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     3601 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     3661 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     3721 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3781 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3841 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3901 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     3961 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4021 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4081 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4141 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     4201 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     4261 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     4321 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     4381 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     4441 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     4501 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     4561 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     4621 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     4681 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     4741 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     4801 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4861 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4921 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4981 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     5041 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     5101 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     5161 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     5221 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     5281 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     5341 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     5401 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     5461 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     5521 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5581 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5641 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     5701 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5761 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5821 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5881 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5941 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     6001 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     6061 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     6121 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     6181 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     6241 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6301 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6361 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6421 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6481 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6541 ggtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6601 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6661 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6721 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6781 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6841 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6901 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6961 cttaccatct g
//
