LOCUS       sequence-430807-        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: GPR1-DuET
FEATURES             Location/Qualifiers
     source          1..6605
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      209..797
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     698..717
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     951..968
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1085..1106
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1121..1126,1127..1144,1145..1151)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1159..1175
                     /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     1164..1186
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1183..1199
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1183..1199
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1248..1369
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1259..1278)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1313..1332
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1543..2337)
                     /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(1654..1673)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2264..2283
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2404..2733)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2418..2553
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2472..2491)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2718..2738
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2747..3175)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2857..2878)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3069..3088
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3221..3445
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3434..3451
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4765..4784)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4766..4784)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4829..5032)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4858..4878)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5033..5412
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5584..5603
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5678..5782
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5783..5851,5852..6605,1..38)
                     /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(6001..6020)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
       61 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
      121 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
      181 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
      241 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
      301 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat accgttcttc
      361 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
      421 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
      481 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
      541 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
      601 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
      661 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
      721 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
      781 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
      841 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
      901 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
      961 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     1021 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     1081 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     1141 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     1201 atgattacgc caagctctag ctagaggtcg acggtataca gacatgataa gatacattga
     1261 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     1321 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg gggtgggcga
     1381 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga
     1441 ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt
     1501 tgggcgtcgc ttggtcggtc atttcgaacc ccagagtccc gctcagaaga actcgtcaag
     1561 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     1621 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
     1681 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     1741 ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc
     1801 gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     1861 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     1921 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     1981 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     2041 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     2101 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag
     2161 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     2221 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     2281 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
     2341 aaacgatcct catcctgtct cttgatcaga tccgaaaatg gatatacaag ctcccgggag
     2401 ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga
     2461 ggcagaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga
     2521 atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt
     2581 tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt
     2641 tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga
     2701 gcctggggac tttccacacc ctaactgaca cacattccac agaattaatt cgcgttaaat
     2761 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     2821 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     2881 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     2941 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3001 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3061 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3121 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtgggga
     3181 taccccctag agccccagct ggttctttcc gcctcagaag ccatagagcc caccgcatcc
     3241 ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc caccccaccc
     3301 cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt ttattaggaa
     3361 aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg gcaaacaaca
     3421 gatggctggc aactagaagg cacagtcgag gctgatcagc gggtttaaac gggccctcta
     3481 gactcgagtt aggcaggggc cacctgagaa gtctcggtct tagacacggt ggtgctggcc
     3541 tcatcgaatt caccggtgcg tccaccggta tcgatctgcg ctgtttccag caagcacaga
     3601 tttttagtct cagagttcct caattgttct gacacagtcc cgctacagga tacttcccaa
     3661 agtgtgtact tcagaatctc agcaacagag gacctaaagc gcgcctggaa ctttttggaa
     3721 atgagaacat acaggattgg attcaggcag ctattcagga aggccaagcc agtgctcagg
     3781 gggatacccg cttgcatcac gtgatgtgag taagagttgt ggtgaatagt cagctcccaa
     3841 atggaaaaca aatggtaggg tgtccaacag acaacgaatg cgacgaccac cacaaggatc
     3901 gtccaaaaat gcctactaga aatcaggatg ctccgcttct tgaccttgaa gataaggcac
     3961 aagtaacaga tggacatagt gagcagaggg aagaggtacc ctatgatgaa tttgacccag
     4021 gtcagtacgt gatgccgaat cagggtaagg tctggatcat gcttctgaaa gttattatag
     4081 cacaatgtgt ggttgttgaa ttccacggtg tcgcgaaaat acagtgctgg cccgccgatc
     4141 agagaagcga ggagccaaat gaagataatg acaatcagac tgtttttcag cgtccgatgc
     4201 cgatgagaca gcaccggatg aatgagatga atataatggt cgaggctaat caccgtcaga
     4261 aagaaaactg aggcaaacat attcagttgg gcagtaaagc tattagcctt gcacagccag
     4321 atgccaaagg gccagtgaaa gttcatggct acatagctga tgtacagagg caggaacagg
     4381 aggaagatga agtcagcgat ggccaagttc aggaaccaaa gggtagttac cgtcttcttc
     4441 cacttaaagc cagtgaacca aatgacgatt gcattgccag gtatacccag gacgaatgcc
     4501 agacagtaga ggacgaggga aacccagtgc accactccca gctgcacctt ctcctcaaga
     4561 tcactttcca gtgagtagta gtccaaatca taactatagt tctcgaattc ctcaaacagt
     4621 gtttcctcca agtcttccat atcgatgctg gcgtcatcat cgtccttgta gtcggcgaat
     4681 accaggcaga agatgtagct cagggcgatg atcgtcttca tggtggcgct aagcttaagt
     4741 ttaaacgcta gccagcttgg gtctccctat agtgagtcgt attaatttcg ataagccagt
     4801 aagcagtggg ttctctagtt agccagagag ctctgcttat atagacctcc caccgtacac
     4861 gcctaccgcc catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt
     4921 gattttggtg ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc
     4981 gtgagtcaaa ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa
     5041 tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact
     5101 gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca
     5161 tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac
     5221 gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg
     5281 ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga
     5341 actccatata tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca
     5401 ataatcaatg tcaacgcgta tatctggccc gtacatcgcg aagcagcgca aaacgcctaa
     5461 ccctaagcag attcttcatg caaatgtcgg tcaagccttg ccttgttgta gcttaaattt
     5521 tgctcgcgca ctactcagcg acctccaaca cacaagcagg gagcagatac tggcttaact
     5581 atgcggcatc agagcagatt gtactgagag tgcaccatag gggatcggga gatctcccga
     5641 tccgtcgacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5701 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5761 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5821 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5881 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5941 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     6001 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     6061 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     6121 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     6181 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6241 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6301 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6361 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6421 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6481 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6541 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6601 acaga
//
