LOCUS       sequence-430768-        6572 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 6572)
  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 6572)
  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: CCRL2-DuET
FEATURES             Location/Qualifiers
     source          1..6572
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      204..792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     693..712
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     946..963
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1080..1101
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1116..1121,1122..1139,1140..1146)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1154..1170
                     /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     1159..1181
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1178..1194
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1178..1194
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1243..1364
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1254..1273)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1308..1327
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1538..2332)
                     /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(1649..1668)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2259..2278
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2399..2728)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2413..2548
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2467..2486)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2713..2733
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2742..3170)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2852..2873)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3064..3083
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3216..3440
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3429..3446
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4727..4746)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4728..4746)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4791..4994)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4820..4840)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4995..5374
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5546..5565
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5640..5744
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5745..5813,5814..6572,1..33)
                     /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(5963..5982)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
       61 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
      121 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
      181 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
      241 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      301 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaataccgt tcttctagtg
      361 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      421 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      481 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      541 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      601 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      661 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      721 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      781 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      841 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
      901 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
      961 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     1021 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     1081 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     1141 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     1201 tacgccaagc tctagctaga ggtcgacggt atacagacat gataagatac attgatgagt
     1261 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     1321 ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg ggcgaagaac
     1381 tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg
     1441 aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc
     1501 gtcgcttggt cggtcatttc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc
     1561 gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt
     1621 cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat
     1681 agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca
     1741 ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca
     1801 tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca
     1861 gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt
     1921 tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat
     1981 cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg
     2041 gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg
     2101 cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat
     2161 tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc
     2221 ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc
     2281 tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg
     2341 atcctcatcc tgtctcttga tcagatccga aaatggatat acaagctccc gggagctttt
     2401 tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggcag
     2461 aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg
     2521 cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg
     2581 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
     2641 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     2701 gggactttcc acaccctaac tgacacacat tccacagaat taattcgcgt taaatttttg
     2761 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2821 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     2881 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     2941 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     3001 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     3061 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     3121 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ggggataccc
     3181 cctagagccc cagctggttc tttccgcctc agaagccata gagcccaccg catccccagc
     3241 atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag
     3301 aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac
     3361 agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg
     3421 ctggcaacta gaaggcacag tcgaggctga tcagcgggtt taaacgggcc ctctagactc
     3481 gagttaggca ggggccacct gagaagtctc ggtcttagac acggtggtgc tggcctcatc
     3541 gaattcaccg gtgcgtccac cggtatcgat cacttctgta ctatggtcgg gctcctcccg
     3601 tgaggtgccc tgggcgctct gacccctggg ctgcaaaggg gtgttagatc tcaggtggaa
     3661 acatctgcac agatacttgg aaaacgtccc gtccaggaag gcgtacagga ggggattgat
     3721 gcaacagtgt gttgtggcaa taagtttggt gatgtggacg gacttgtcca ggttgtagct
     3781 gctcttgcag tcgctcagtg aaaaatgctc cttaaaagtg gaaaggaaga acgcaatatt
     3841 atagggtgcc cacatcagca agaacacgac cattattgca aacaccagct taaacagaga
     3901 atatcgctgc tctctaaatc gaagtgtctt tctcatttgc acatacagaa atgtaaagat
     3961 gaacagtggc aacaccagta cggaaatatt cattttaagg gtaagaaagt gcttccagaa
     4021 ggtctcgtct gccggaagga aaggtgtccg agaaaaggcg catttgtact tttgatcctc
     4081 catttgaggc ttatagacca cgaactcggg cagggttgcc aggatcgccg taacccacgc
     4141 caaaacgctt gtaattatac cgcacggcac tcgtctcctg gcagagaaaa agttaccttt
     4201 gtgcaggaaa accaggtacc gttggacggt gagcaagcaa ttgaaaaacg tctcagaata
     4261 aagtcccacg aagtacagtc ctatcaaaat cttacacatt gggtcgcccc cagcgtgagc
     4321 ccagaatggc aaggtcagca gaaaacacag attggacaca gccagattca gcaggtaaat
     4381 gttctcgacg cgcttcaggc ccttgtactt cacaagtatg agcaccacca acagattgtc
     4441 caggactccg atcacaaaga cggctgaaca gaggctgggg acaagctgag cggagagagc
     4501 ctgtgcgtca tacttatcac attgttcggc ttcgtcgctt tccagttcac cctcaatcag
     4561 cacatcgtat tcatcctcag gcgccagcgt ataattggcc atatcgatgc tggcgtcatc
     4621 atcgtccttg tagtcggcga ataccaggca gaagatgtag ctcagggcga tgatcgtctt
     4681 catggtggcg ctaagcttaa gtttaaacgc tagccagctt gggtctccct atagtgagtc
     4741 gtattaattt cgataagcca gtaagcagtg ggttctctag ttagccagag agctctgctt
     4801 atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta
     4861 cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg
     4921 gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc
     4981 caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga
     5041 aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
     5101 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
     5161 cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca
     5221 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
     5281 accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt
     5341 gattactatt aataactagt caataatcaa tgtcaacgcg tatatctggc ccgtacatcg
     5401 cgaagcagcg caaaacgcct aaccctaagc agattcttca tgcaaatgtc ggtcaagcct
     5461 tgccttgttg tagcttaaat tttgctcgcg cactactcag cgacctccaa cacacaagca
     5521 gggagcagat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     5581 aggggatcgg gagatctccc gatccgtcga cgtcaggtgg cacttttcgg ggaaatgtgc
     5641 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5701 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5761 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5821 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5881 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5941 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6001 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     6061 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     6121 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6181 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     6241 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     6301 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6361 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     6421 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6481 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6541 caactatgga tgaacgaaat agacagatcg ct
//
