LOCUS       sequence-430774-        6653 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 6653)
  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 6653)
  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: CMKLR1-DuET
FEATURES             Location/Qualifiers
     source          1..6653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     491..510
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     744..761
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    878..899
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(914..919,920..937,938..944)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    952..968
                     /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     957..979
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     976..992
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     976..992
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1041..1162
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1052..1071)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1106..1125
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1336..2130)
                     /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(1447..1466)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2057..2076
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2197..2526)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2211..2346
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2265..2284)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2511..2531
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2540..2968)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2650..2671)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2862..2881
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3014..3238
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3227..3244
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4606..4625)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4607..4625)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4670..4873)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4699..4719)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4874..5253
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5425..5444
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5519..5623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5624..5692,5693..6484)
                     /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(5842..5861)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
       61 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
      121 tcagcagagc gcagatacca aataccgttc ttctagtgta gccgtagtta ggccaccact
      181 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
      241 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
      301 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
      361 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
      421 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
      481 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      541 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      601 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
      661 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
      721 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
      781 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
      841 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
      901 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
      961 gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc tagctagagg
     1021 tcgacggtat acagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     1081 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     1141 tataagctgc aataaacaag ttggggtggg cgaagaactc cagcatgaga tccccgcgct
     1201 ggaggatcat ccagccggcg tcccggaaaa cgattccgaa gcccaacctt tcatagaagg
     1261 cggcggtgga atcgaaatct cgtgatggca ggttgggcgt cgcttggtcg gtcatttcga
     1321 accccagagt cccgctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga
     1381 atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc
     1441 ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg
     1501 gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc
     1561 atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa
     1621 cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc
     1681 ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca
     1741 ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc
     1801 ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca
     1861 gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc
     1921 cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt
     1981 cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca
     2041 gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga
     2101 acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc
     2161 agatccgaaa atggatatac aagctcccgg gagctttttg caaaagccta ggcctccaaa
     2221 aaagcctcct cactacttct ggaatagctc agaggcagag gcggcctcgg cctctgcata
     2281 aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg
     2341 cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg
     2401 catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag
     2461 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg
     2521 acacacattc cacagaatta attcgcgtta aatttttgtt aaatcagctc attttttaac
     2581 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     2641 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     2701 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     2761 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     2821 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     2881 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     2941 gcgcttaatg cgccgctaca gggcgcgtgg ggataccccc tagagcccca gctggttctt
     3001 tccgcctcag aagccataga gcccaccgca tccccagcat gcctgctatt gtcttcccaa
     3061 tcctccccct tgctgtcctg ccccacccca ccccccagaa tagaatgaca cctactcaga
     3121 caatgcgatg caatttcctc attttattag gaaaggacag tgggagtggc accttccagg
     3181 gtcaaggaag gcacggggga ggggcaaaca acagatggct ggcaactaga aggcacagtc
     3241 gaggctgatc agcgggttta aacgggccct ctagactcga gttaggcagg ggccacctga
     3301 gaagtctcgg tcttagacac ggtggtgctg gcctcatcga attcaccggt gcgtccaccg
     3361 gtatcgataa gcatgccggt ctcgcgttca ttcatgcttg tgcgctcgtt cattgaagac
     3421 atcttggtaa aagagcgatg gctaggataa ctggaatgac ctgtatcctc ggacagtgcg
     3481 ttaacgaggc gagagaacag ggccaccttg aactttttaa agtcctgtcc cataaacacg
     3541 tacaaaatag gattcatgca actattagcg atcgcgagag cggtggccaa tgggagtccc
     3601 aggctgaaga cagagccggg catggccgtg tggtgcagct ccagaagatt caaagtatga
     3661 tatggacacc aacacagaaa gaacgtaatg ataatcgtga cgatgatctt gaatggcttc
     3721 ttagtcttag caagccgatt cctttgcagc ttacagacga tggtcaggta acatgccgtt
     3781 ataatgagaa ctggcaccag gaacccgcag aggaaccgcg tcacggtcac taccatgtgc
     3841 cgggaatagc ctacgggatc catctggctg tgtgttggcc aagaactgct gcccggggtg
     3901 gacaagctga agttgttgaa gcaggaaatc ttaccgtgga gattcgctgt gtcccgaaag
     3961 accaggctag ggctgctcag aaaaaatgcc agcacccaga tcaccataca tgccatataa
     4021 gccagcctaa cgcttctgtg attctgagac cacactggaa gcagcacaga aatgcaccga
     4081 tcggaggaga taatggtcag gagaaaaaca gaggtaaaca tgttatgtat caagaggaaa
     4141 ttggagattt tgcacatggc ggtaccaaag acccaatggt agtccattgc ggcataggta
     4201 atgtggatag gcagaaacac attgaagagg aagtccgcca cagccaggtt cagaaaccac
     4261 accatgttga cagtcttctt catcttgaat gtggctatta tgataacaag tccgttgccc
     4321 aggatgccca ggaaacaaac gatgctatag accactacca gaaaaattct ggttacgcgt
     4381 gcctccagag ggctcagatc ttccagcacc acgatagaat ccaggtagtc gggatattcg
     4441 tccccataag agatgctggt gttgtagtct tcgtcttcca tatcgatgct ggcgtcatca
     4501 tcgtccttgt agtcggcgaa taccaggcag aagatgtagc tcagggcgat gatcgtcttc
     4561 atggtggcgc taagcttaag tttaaacgct agccagcttg ggtctcccta tagtgagtcg
     4621 tattaatttc gataagccag taagcagtgg gttctctagt tagccagaga gctctgctta
     4681 tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac
     4741 gacattttgg aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg
     4801 ggtggagact tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc
     4861 aaaaccgcat caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa
     4921 agtcccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt
     4981 caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc
     5041 gtaaatactc cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat
     5101 acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta
     5161 ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac cccgtaattg
     5221 attactatta ataactagtc aataatcaat gtcaacgcgt atatctggcc cgtacatcgc
     5281 gaagcagcgc aaaacgccta accctaagca gattcttcat gcaaatgtcg gtcaagcctt
     5341 gccttgttgt agcttaaatt ttgctcgcgc actactcagc gacctccaac acacaagcag
     5401 ggagcagata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     5461 ggggatcggg agatctcccg atccgtcgac gtcaggtggc acttttcggg gaaatgtgcg
     5521 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5581 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5641 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5701 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5761 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5821 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5881 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5941 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6001 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6061 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6121 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6181 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6241 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6301 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6361 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6421 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6481 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6541 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6601 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat ctt
//
