LOCUS       sequence-430763-        6620 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 6620)
  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 6620)
  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: CCR4-DuET
FEATURES             Location/Qualifiers
     source          1..6620
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     580..599
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(818..922)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(997..1016)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1188..1567
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1568..1771
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1722..1742
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1816..1835
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1816..1834
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3164..3181)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3170..3394
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3440..3868
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3527..3546)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3737..3758
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3877..3897)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3882..4211
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4062..4197
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4124..4143
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4278..5072
                     /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(4332..4351)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4942..4961
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5246..5367
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5283..5302)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5337..5356
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5416..5432)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5416..5432)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5429..5451)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5440..5456
                     /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(5464..5470,5471..5488,5489..5494))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5509..5530
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5647..5664)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5818..6406)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5898..5917)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6577..6620,1..748,749..817))
                     /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 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
       61 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
      121 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
      181 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
      241 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
      301 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
      361 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
      421 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
      481 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
      541 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
      601 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
      661 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
      721 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
      781 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
      841 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
      901 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
      961 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     1021 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     1081 aatttaagct acaacaaggc aaggcttgac cgacatttgc atgaagaatc tgcttagggt
     1141 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
     1201 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     1261 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     1321 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     1381 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     1441 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     1501 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     1561 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     1621 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     1681 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     1741 tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac ccactgctta
     1801 ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct agcgtttaaa
     1861 cttaagctta gcgccaccat gaagacgatc atcgccctga gctacatctt ctgcctggta
     1921 ttcgccgact acaaggacga tgatgacgcc agcatcgata tgaatcccac tgacatcgca
     1981 gataccactt tggacgagag tatctacagc aattactact tgtacgaaag catcccaaag
     2041 ccgtgcacta aggagggcat taaagcgttc ggggagcttt tcctcccccc cttgtactcc
     2101 ctggtgtttg tcttcggctt gttgggtaac tccgttgtgg tattggtatt gttcaagtac
     2161 aagagactta ggagcatgac cgacgtttac ttgctgaatc tggcaattag cgatcttctt
     2221 ttcgtatttt ccctgccctt ttgggggtat tatgcggccg accagtgggt gttcggactc
     2281 ggcctgtgta agatgatttc ttggatgtac cttgtggggt tctactcagg gatctttttc
     2341 gtgatgctca tgtctattga tagatatttg gccattgtgc atgctgtctt ttctcttcgg
     2401 gcaaggactc tcacctatgg ggttattacc tccctggcca cgtggtcagt ggcagttttc
     2461 gcctcactcc ccggattcct cttcagcacg tgctacacag agcgcaacca tacgtattgt
     2521 aagaccaagt acagcctcaa tagcacaaca tggaaggtgc tttctagtct ggagattaat
     2581 atcctggggc ttgtgattcc gctgggcatc atgctctttt gctacagcat gataattaga
     2641 acactgcagc attgtaaaaa cgagaaaaaa aacaaggcgg tcaagatgat cttcgcagtg
     2701 gtagtactgt tcctggggtt ctggacacct tataacatag tactcttttt ggaaacactg
     2761 gtagaactcg aagtactgca ggattgcacc ttcgagcgct acctggatta cgccatccag
     2821 gctaccgaga cacttgcctt cgtacactgt tgtcttaatc caattatcta ctttttcctt
     2881 ggcgaaaagt tcagaaagta cattttgcag ctctttaaga cctgccgggg tctctttgtg
     2941 ttgtgtcaat attgcgggct tctccagatc tacagcgcgg acacaccctc aagctcttat
     3001 acacagagta caatggacca tgacctgcat gacgccctca tcgataccgg tggacgcacc
     3061 ggtgaattcg atgaggccag caccaccgtg tctaagaccg agacttctca ggtggcccct
     3121 gcctaactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     3181 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3241 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3301 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3361 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     3421 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3481 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3541 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3601 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3661 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3721 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3781 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3841 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     3901 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3961 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4021 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     4081 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4141 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4201 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     4261 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     4321 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4381 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4441 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4501 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4561 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4621 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4681 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4741 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4801 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4861 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4921 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4981 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     5041 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     5101 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     5161 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     5221 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     5281 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     5341 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     5401 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5461 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5521 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5581 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5641 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5701 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5761 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5821 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5881 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5941 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6001 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6061 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6121 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6181 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6241 ctacggctac actagaagaa cggtatttgg tatctgcgct ctgctgaagc cagttacctt
     6301 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6361 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6421 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6481 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6541 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6601 acctatctca gcgatctgtc
//
