LOCUS       sequence-430767-        6605 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 6605)
  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 6605)
  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: CCR8-DuET
FEATURES             Location/Qualifiers
     source          1..6605
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      190..778
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     679..698
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     932..949
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1066..1087
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1102..1107,1108..1125,1126..1132)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1140..1156
                     /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     1145..1167
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1164..1180
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1164..1180
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1229..1350
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1240..1259)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1294..1313
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1524..2318)
                     /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(1635..1654)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2245..2264
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2385..2714)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2399..2534
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2453..2472)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2699..2719
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2728..3156)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2838..2859)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3050..3069
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3202..3426
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3415..3432
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4746..4765)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4747..4765)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4810..5013)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4839..4859)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5014..5393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5565..5584
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5659..5763
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5764..5832,5833..6605,1..19)
                     /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(5982..6001)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
       61 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
      121 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
      181 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
      241 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
      301 aactggcttc agcagagcgc agataccaaa taccgttctt ctagtgtagc cgtagttagg
      361 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
      421 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
      481 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
      541 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
      601 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
      661 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
      721 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
      781 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
      841 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
      901 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      961 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     1021 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     1081 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     1141 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcta
     1201 gctagaggtc gacggtatac agacatgata agatacattg atgagtttgg acaaaccaca
     1261 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     1321 gtaaccatta taagctgcaa taaacaagtt ggggtgggcg aagaactcca gcatgagatc
     1381 cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg attccgaagc ccaacctttc
     1441 atagaaggcg gcggtggaat cgaaatctcg tgatggcagg ttgggcgtcg cttggtcggt
     1501 catttcgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg
     1561 cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg
     1621 ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca
     1681 cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc
     1741 aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc
     1801 ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg
     1861 acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg
     1921 aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat
     1981 actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat
     2041 agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc
     2101 gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac
     2161 aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca
     2221 tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg
     2281 gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc
     2341 tcttgatcag atccgaaaat ggatatacaa gctcccggga gctttttgca aaagcctagg
     2401 cctccaaaaa agcctcctca ctacttctgg aatagctcag aggcagaggc ggcctcggcc
     2461 tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga
     2521 gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc
     2581 atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac
     2641 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     2701 cctaactgac acacattcca cagaattaat tcgcgttaaa tttttgttaa atcagctcat
     2761 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     2821 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     2881 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     2941 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     3001 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     3061 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     3121 cacccgccgc gcttaatgcg ccgctacagg gcgcgtgggg atacccccta gagccccagc
     3181 tggttctttc cgcctcagaa gccatagagc ccaccgcatc cccagcatgc ctgctattgt
     3241 cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc
     3301 tactcagaca atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac
     3361 cttccagggt caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag
     3421 gcacagtcga ggctgatcag cgggtttaaa cgggccctct agactcgagt taggcagggg
     3481 ccacctgaga agtctcggtc ttagacacgg tggtgctggc ctcatcgaat tcaccggtgc
     3541 gtccaccggt atcgatcaga atgtaatcaa cggaactaga tctgctactg tgctgctggc
     3601 aactgcttga cttttcacaa ctttctctgg gcatctgcct ccccagatag ttgaagatct
     3661 gtgaacaaga cttttgaaaa atctcgctga ggtgtttttt gaatttctcg cccacaaatg
     3721 catagatcac agggttcacg cagcaatgtg taaaactaat aatctcggtc acgtgtgtag
     3781 cgtaagtcaa ctgctgtgaa atgctacagc catccaaaat atgcattgaa tgcaggcttg
     3841 tcaagaacag cactacgttg aaaggcaccc agaacaacag tgaggctatc acgacaatca
     3901 gcaccaacct gatagccttg gtcttattat gattctggca gcgtttcagt tggtgcagaa
     3961 tcttaatgta gcaaaacata aagatagtga aaggaatcag gaggccgaga atgttcatct
     4021 tgaagttggt aaagatcttc cacttgagag tctgttgatt ataaaaactg tagcactgaa
     4081 gcactccgtc ctcacttgca acctggtaaa acaccagaag cgggatggtc gccataatag
     4141 cggtcaacca gacggccagg caaagtgttg tccccatgcg gatagttctg accttcagag
     4201 cgtaaacggc atggactacg gccaggtacc tatccactga catcagcgtg atgaagaaca
     4261 tggaagaata gaatccgata tagtaaaagc cggacacgac cttgcacatt acggtcccga
     4321 atacccactg atcgagcagg tagtatgtct ggaacgggaa actaaagaca aacaacagat
     4381 cgctcagggc caggttcagc agatacacat cggtaatact cctgagtttt ttgcacacta
     4441 ccagcaccag tatcaccaag ctgttgccca gcaggctgaa cacgaacagc aggcagtaga
     4501 aaactgccaa cagaagtttg ccgttagttt gaatcaactc agcgtcacaa gggctgctga
     4561 agatgtcggg gtaataatag tctgttacag tcgtcactga caggtcgaga gtatagtcca
     4621 tatcgatgct ggcgtcatca tcgtccttgt agtcggcgaa taccaggcag aagatgtagc
     4681 tcagggcgat gatcgtcttc atggtggcgc taagcttaag tttaaacgct agccagcttg
     4741 ggtctcccta tagtgagtcg tattaatttc gataagccag taagcagtgg gttctctagt
     4801 tagccagaga gctctgctta tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt
     4861 caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt gccaaaacaa
     4921 actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa accgctatcc
     4981 acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga ctaatacgta
     5041 gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat gccaggcggg
     5101 ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac
     5161 tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtccctat
     5221 tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg
     5281 tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat atgggctatg
     5341 aactaatgac cccgtaattg attactatta ataactagtc aataatcaat gtcaacgcgt
     5401 atatctggcc cgtacatcgc gaagcagcgc aaaacgccta accctaagca gattcttcat
     5461 gcaaatgtcg gtcaagcctt gccttgttgt agcttaaatt ttgctcgcgc actactcagc
     5521 gacctccaac acacaagcag ggagcagata ctggcttaac tatgcggcat cagagcagat
     5581 tgtactgaga gtgcaccata ggggatcggg agatctcccg atccgtcgac gtcaggtggc
     5641 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5701 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5761 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5821 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5881 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5941 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     6001 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     6061 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6121 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6181 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6241 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6301 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6361 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6421 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6481 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6541 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6601 gcctc
//
