LOCUS       sequence-430757-        6590 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 6590)
  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 6590)
  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: C5A-DuET
FEATURES             Location/Qualifiers
     source          1..6590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      73..661
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     562..581
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     815..832
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    949..970
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(985..990,991..1008,1009..1015)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1023..1039
                     /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     1028..1050
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1047..1063
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1047..1063
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1112..1233
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1123..1142)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1177..1196
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1407..2201)
                     /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(1518..1537)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2128..2147
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2268..2597)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2282..2417
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2336..2355)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2582..2602
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2611..3039)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2721..2742)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2933..2952
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3085..3309
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3298..3315
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4614..4633)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4615..4633)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4678..4881)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4707..4727)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4882..5261
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5433..5452
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5527..5631
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5632..5700,5701..6492)
                     /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(5850..5869)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
       61 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
      121 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
      181 ggtaactggc ttcagcagag cgcagatacc aaataccgtt cttctagtgt agccgtagtt
      241 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
      301 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
      361 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
      421 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
      481 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
      541 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
      601 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
      661 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
      721 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
      781 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
      841 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
      901 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
      961 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     1021 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     1081 ctagctagag gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc
     1141 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     1201 tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag
     1261 atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct
     1321 ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc
     1381 ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
     1441 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     1501 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     1561 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     1621 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     1681 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     1741 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     1801 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     1861 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     1921 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     1981 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     2041 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     2101 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     2161 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     2221 gtctcttgat cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct
     2281 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg
     2341 gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
     2401 ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga
     2461 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct
     2521 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     2581 caccctaact gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct
     2641 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     2701 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     2761 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     2821 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2881 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2941 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     3001 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc
     3061 agctggttct ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat
     3121 tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac
     3181 acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg
     3241 caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag
     3301 aaggcacagt cgaggctgat cagcgggttt aaacgggccc tctagactcg agttaggcag
     3361 gggccacctg agaagtctcg gtcttagaca cggtggtgct ggcctcatcg aattcaccgg
     3421 tgcgtccacc ggtatcgatc acagcctgtg ttttctgtgc catagtgtcg actgtagagc
     3481 gcgtgaaaga tttactctcc ctcaccacac tctcctctgt aagcacgttg cgcagcaaag
     3541 agggcaagga tttccggagt ctgccctgaa acccttgccc agcaacaacg taaatgatag
     3601 gattgataca gcagtttata taggcgaaac tcacacaaag gctgtccaac ttattcaaca
     3661 acaggaaggt tggggagctg ggctccaaaa aggacatcat aataccagtc acctgataag
     3721 gcagccaaaa tataaagaag cttgccacga cggctactac cactttcagt gtctttgtgc
     3781 tccgggtagc tctccttgac caggttctca gcaatataaa tgtgtaacaa attgtgagcg
     3841 tcagcaaggg ccacaaaaag ccaagcacga gccgcacgat tgccacagcc ctctctctcc
     3901 gcttgtcatg gctgtagtcc acaccgcaaa gcacctttgg tgggaagtat tcctccctaa
     3961 caactctgta aaggaatgac gggatagtga ggagcaatgc cagaccccat gcgaccgcgc
     4021 atgcaatcca ggcgaggccc gccccccgga aattttggca ccaaatgggc ttgaacacca
     4081 agagaaacct gtccgcgcta atggttgcga gaaggagtat tgacgcgtac atgttgagca
     4141 gaatcagaga gggcaggata ctacaggcgg ctcctccaaa gggccaatgg tggtgctgca
     4201 caatagatgt gaacaggatg ggcaaggcaa gacaggacag aaagtcggcc actgcgagat
     4261 tcagaaacca aattgcgttg atggtccgtt tagcttcaaa tgctgtaacc cacacgacca
     4321 aggcgtttcc cagcacccca accaggaaca ccacggcgaa aatgaccaat gccaagatat
     4381 cgggaaccct cagcgtgttg cttgtcttat cgacaggggt attaaggtcg agggtgtcct
     4441 tatcgtcata gtgcccgtaa tcaggggtag tatagttaaa tgaattcata tcgatgctgg
     4501 cgtcatcatc gtccttgtag tcggcgaata ccaggcagaa gatgtagctc agggcgatga
     4561 tcgtcttcat ggtggcgcta agcttaagtt taaacgctag ccagcttggg tctccctata
     4621 gtgagtcgta ttaatttcga taagccagta agcagtgggt tctctagtta gccagagagc
     4681 tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca atggggcgga
     4741 gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac tcccattgac
     4801 gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac gcccattgat
     4861 gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga tgtactgcca
     4921 agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc
     4981 attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc
     5041 agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg gcgttactat
     5101 gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg
     5161 gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc
     5221 cgtaattgat tactattaat aactagtcaa taatcaatgt caacgcgtat atctggcccg
     5281 tacatcgcga agcagcgcaa aacgcctaac cctaagcaga ttcttcatgc aaatgtcggt
     5341 caagccttgc cttgttgtag cttaaatttt gctcgcgcac tactcagcga cctccaacac
     5401 acaagcaggg agcagatact ggcttaacta tgcggcatca gagcagattg tactgagagt
     5461 gcaccatagg ggatcgggag atctcccgat ccgtcgacgt caggtggcac ttttcgggga
     5521 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5581 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5641 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5701 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5761 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5821 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5881 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5941 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     6001 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     6061 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     6121 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6181 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6241 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6301 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     6361 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6421 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6481 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6541 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
//
