LOCUS       sequence-430800-        6644 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 6644)
  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 6644)
  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: GAL3-DuET
FEATURES             Location/Qualifiers
     source          1..6644
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      228..816
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     717..736
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     970..987
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1104..1125
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1140..1145,1146..1163,1164..1170)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1178..1194
                     /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     1183..1205
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1202..1218
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1202..1218
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1267..1388
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1278..1297)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1332..1351
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1562..2356)
                     /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(1673..1692)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2283..2302
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2423..2752)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2437..2572
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2491..2510)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2737..2757
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2766..3194)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2876..2897)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3088..3107
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3240..3464
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3453..3470
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4823..4842)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4824..4842)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4887..5090)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4916..4936)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5091..5470
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5642..5661
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5736..5840
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5841..5909,5910..6644,1..57)
                     /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(6059..6078)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
       61 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
      121 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
      181 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
      241 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
      301 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
      361 ataccaaata ccgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
      421 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
      481 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
      541 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
      601 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
      661 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
      721 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
      781 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
      841 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
      901 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
      961 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     1021 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     1081 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     1141 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     1201 acaggaaaca gctatgacca tgattacgcc aagctctagc tagaggtcga cggtatacag
     1261 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     1321 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     1381 aacaagttgg ggtgggcgaa gaactccagc atgagatccc cgcgctggag gatcatccag
     1441 ccggcgtccc ggaaaacgat tccgaagccc aacctttcat agaaggcggc ggtggaatcg
     1501 aaatctcgtg atggcaggtt gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg
     1561 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     1621 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
     1681 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     1741 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca
     1801 cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg
     1861 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
     1921 tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
     1981 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
     2041 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
     2101 cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
     2161 gcgctgcctc gtcctgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
     2221 ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt
     2281 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
     2341 catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagat ccgaaaatgg
     2401 atatacaagc tcccgggagc tttttgcaaa agcctaggcc tccaaaaaag cctcctcact
     2461 acttctggaa tagctcagag gcagaggcgg cctcggcctc tgcataaata aaaaaaatta
     2521 gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag
     2581 ttaggggcgg gactatggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
     2641 ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc atgctttgca
     2701 tacttctgcc tgctggggag cctggggact ttccacaccc taactgacac acattccaca
     2761 gaattaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     2821 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     2881 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     2941 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     3001 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     3061 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     3121 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     3181 gctacagggc gcgtggggat accccctaga gccccagctg gttctttccg cctcagaagc
     3241 catagagccc accgcatccc cagcatgcct gctattgtct tcccaatcct cccccttgct
     3301 gtcctgcccc accccacccc ccagaataga atgacaccta ctcagacaat gcgatgcaat
     3361 ttcctcattt tattaggaaa ggacagtggg agtggcacct tccagggtca aggaaggcac
     3421 gggggagggg caaacaacag atggctggca actagaaggc acagtcgagg ctgatcagcg
     3481 ggtttaaacg ggccctctag actcgagtta ggcaggggcc acctgagaag tctcggtctt
     3541 agacacggtg gtgctggcct catcgaattc accggtgcgt ccaccggtat cgatctcggg
     3601 ccccctcgct gcctcccctc cgtgcaccgg gccctcgcgc ggctctgggc cttggccacc
     3661 tccggccaac agtctcccgc tgggccgtgc atcgccagga catcctggag gaccagagct
     3721 cgcgggtctc acccgtctga gggctcggcg ggctctgtgc ctccttcgtc ttccgcaagg
     3781 ccagagccgc cggaatcggg cgcgaaagtg gcgggaagcc aaggcgtaca ccaatgggtt
     3841 caggcaagaa ttagcatacg ccaggcagtg agatgccagt ctgcaagcgt atgtggcggg
     3901 agagaaggcg aatctgccat accagaagca caggatcagg gcgtgatgag ggccccagca
     3961 gagcgcgtac aaggccgcca cggccagcat tgccctccca gctcggcctg tagcgcgcct
     4021 tcttgcctcg gcggctgcgg cccctgccgg ccctacggca gcccagagga accgcagcgt
     4081 gcgcccgtat gcgaggctga ccacagctac gggcagcaag tagccagcgg caaaggtagc
     4141 cacgtccaat gcccgtctcc gggcatcttc ccaggcgggc acgcacagct caagtgcccc
     4201 gtacctgaca gtcccatagt atgacagata tggtgcactg aacaacgcgg ccaacagcca
     4261 gaccagtccc acggcggccc gtgcgttccg aggtgttcgg agtgcgcgag atcgaagcgg
     4321 atgtctcact gccaggtagc ggtccacgct cacagcagcc aaggtaaatg atgatgcgta
     4381 catcgtgagg tagataagca gatgcacagc cttgcagacc aaggcaccga acagccaagc
     4441 gtcgagggta taaattgtag cttgaaacgg gacgcaacac aagatgaagc acagatctgc
     4501 tacagccaaa ttcaggatga acagatccgt ggttgacccc ggctcttgcc acgcactagg
     4561 cccaggttgc agcaggacgg ccaggaccag tccgtttcca acggtgccca gcaggaatat
     4621 gagggcaaaa accacgggga cagcgactgc tcccacgctg cctggggagt ccaggctgat
     4681 attctgtgcg tcagccatat cgatgctggc gtcatcatcg tccttgtagt cggcgaatac
     4741 caggcagaag atgtagctca gggcgatgat cgtcttcatg gtggcgctaa gcttaagttt
     4801 aaacgctagc cagcttgggt ctccctatag tgagtcgtat taatttcgat aagccagtaa
     4861 gcagtgggtt ctctagttag ccagagagct ctgcttatat agacctccca ccgtacacgc
     4921 ctaccgccca tttgcgtcaa tggggcggag ttgttacgac attttggaaa gtcccgttga
     4981 ttttggtgcc aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt
     5041 gagtcaaacc gctatccacg cccattgatg tactgccaaa accgcatcac catggtaata
     5101 gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg
     5161 gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata
     5221 tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt
     5281 caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg
     5341 cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac
     5401 tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat
     5461 aatcaatgtc aacgcgtata tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc
     5521 ctaagcagat tcttcatgca aatgtcggtc aagccttgcc ttgttgtagc ttaaattttg
     5581 ctcgcgcact actcagcgac ctccaacaca caagcaggga gcagatactg gcttaactat
     5641 gcggcatcag agcagattgt actgagagtg caccataggg gatcgggaga tctcccgatc
     5701 cgtcgacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5761 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5821 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5881 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5941 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     6001 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6061 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6121 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6181 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6241 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6301 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6361 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6421 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6481 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6541 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6601 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatg
//
