LOCUS       sequence-430829-        6626 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 6626)
  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 6626)
  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: GPR4-DuET
FEATURES             Location/Qualifiers
     source          1..6626
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      116..704
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     605..624
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     858..875
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    992..1013
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1028..1033,1034..1051,1052..1058)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1066..1082
                     /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     1071..1093
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1090..1106
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1090..1106
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1155..1276
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1166..1185)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1220..1239
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1450..2244)
                     /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(1561..1580)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2171..2190
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2311..2640)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2325..2460
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2379..2398)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2625..2645
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2654..3082)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2764..2785)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2976..2995
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3128..3352
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3341..3358
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4693..4712)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4694..4712)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4757..4960)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4786..4806)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4961..5340
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5512..5531
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5606..5710
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5711..5779,5780..6571)
                     /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(5929..5948)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
       61 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
      121 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
      181 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
      241 gagcgcagat accaaatacc gttcttctag tgtagccgta gttaggccac cacttcaaga
      301 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
      361 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
      421 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
      481 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
      541 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
      601 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
      661 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
      721 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
      781 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
      841 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
      901 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
      961 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     1021 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     1081 aatttcacac aggaaacagc tatgaccatg attacgccaa gctctagcta gaggtcgacg
     1141 gtatacagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     1201 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     1261 ctgcaataaa caagttgggg tgggcgaaga actccagcat gagatccccg cgctggagga
     1321 tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag aaggcggcgg
     1381 tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca
     1441 gagtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg
     1501 agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc
     1561 aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca
     1621 gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc
     1681 atgggtcacg acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc
     1741 ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc
     1801 catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc
     1861 cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg
     1921 agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct
     1981 tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca
     2041 cgatagccgc gctgcctcgt cctgcagttc attcagggca ccggacaggt cggtcttgac
     2101 aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat
     2161 tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc
     2221 gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagatcc
     2281 gaaaatggat atacaagctc ccgggagctt tttgcaaaag cctaggcctc caaaaaagcc
     2341 tcctcactac ttctggaata gctcagaggc agaggcggcc tcggcctctg cataaataaa
     2401 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat
     2461 gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact
     2521 tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat
     2581 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac
     2641 attccacaga attaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     2701 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     2761 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     2821 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     2881 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     2941 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     3001 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     3061 aatgcgccgc tacagggcgc gtggggatac cccctagagc cccagctggt tctttccgcc
     3121 tcagaagcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc
     3181 cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact cagacaatgc
     3241 gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc cagggtcaag
     3301 gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac agtcgaggct
     3361 gatcagcggg tttaaacggg ccctctagac tcgagttagg caggggccac ctgagaagtc
     3421 tcggtcttag acacggtggt gctggcctca tcgaattcac cggtgcgtcc accggtatcg
     3481 atctgagctg ggggcagcat cttcaattgc acctggtcgc cctgtgaggg aggggtggcg
     3541 gcccatgacc ctgtcatagc tttcgcggtg gaattccgct tacttgtcaa aggtgtctcc
     3601 aatgtcagag aagcattggc catctcctgt ggtttatcag aggccaggaa tctgagcagg
     3661 ttgtgaagag cctttgcaac atcactcctt gcgccctcgt tcaccaagca gtacaaaata
     3721 gggtcggcaa cgcaattcaa gctggtaaat gccagtgagg aatgataggc tgagaacacc
     3781 cgctcctcga atccgcaatc ccaagggcgg cccagataga tggcggacct gctcagcagc
     3841 aggacgtggt agggagcaaa gcagacaagg acgatggcga tcagggacag cgcaagccgt
     3901 ttaatcttgg ctttttcctg tctttctgta ctaacggaac ctctcacagc cctcaggatg
     3961 ccccggtaag agagcaacat aagagcccat gggaacagga agcccacgaa gaccctgtag
     4021 aggttcatcc aggctaccca cccttccatg gggaactttt cgaaacaaaa tgtgtgattg
     4081 tatcggtccc ggaacaactc gtcatgaaac aggggtgcgg aatttgcgcc caactctgtg
     4141 gcccaaacca cgcttgatac cgccactgca gtcttaactc gcctgagtcg agcgaaccgg
     4201 agtgggtgcg ccacggccag gtaccgatcc acgctgatac agcagagaaa agcgatacta
     4261 atataaatat ttgtgtagaa aatgaaacca aaaagcttac aagaccctgg accgtggatc
     4321 caattgtcat ggtgaaggaa gtagtcgacc caaagaggca gtgtgcagat gtagaggaga
     4381 tcggcaatag acagattcat aaggtacacg cccagttcgt ttctttgctg cacctgtcga
     4441 taggctgccc acagtgcaag gcagtttgtg ggcaagccca ccccgatgac aaaaatgtac
     4501 agggagggtg ggaaaagatg gtccacccgt gaatccacgt ggcacccttc ccaggtatgg
     4561 ttgcccatat cgatgctggc gtcatcatcg tccttgtagt cggcgaatac caggcagaag
     4621 atgtagctca gggcgatgat cgtcttcatg gtggcgctaa gcttaagttt aaacgctagc
     4681 cagcttgggt ctccctatag tgagtcgtat taatttcgat aagccagtaa gcagtgggtt
     4741 ctctagttag ccagagagct ctgcttatat agacctccca ccgtacacgc ctaccgccca
     4801 tttgcgtcaa tggggcggag ttgttacgac attttggaaa gtcccgttga ttttggtgcc
     4861 aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt gagtcaaacc
     4921 gctatccacg cccattgatg tactgccaaa accgcatcac catggtaata gcgatgacta
     4981 atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc
     5041 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt
     5101 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
     5161 tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt
     5221 tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg
     5281 ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc
     5341 aacgcgtata tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat
     5401 tcttcatgca aatgtcggtc aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact
     5461 actcagcgac ctccaacaca caagcaggga gcagatactg gcttaactat gcggcatcag
     5521 agcagattgt actgagagtg caccataggg gatcgggaga tctcccgatc cgtcgacgtc
     5581 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5641 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5701 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5761 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5821 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5881 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5941 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     6001 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     6061 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     6121 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6181 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6241 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6301 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6361 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6421 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6481 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6541 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6601 ttagattgat ttaaaacttc attttt
//
