LOCUS       sequence-430804-        6524 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 6524)
  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 6524)
  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: GNRHR-DuET
FEATURES             Location/Qualifiers
     source          1..6524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      40..628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     529..548
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     782..799
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    916..937
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(952..957,958..975,976..982)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    990..1006
                     /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     995..1017
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1014..1030
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1014..1030
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1079..1200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1090..1109)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1144..1163
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1374..2168)
                     /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(1485..1504)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2095..2114
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2235..2564)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2249..2384
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2303..2322)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2549..2569
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2578..3006)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2688..2709)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2900..2919
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3052..3276
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3265..3282
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4515..4534)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4516..4534)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4579..4782)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4608..4628)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4783..5162
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5334..5353
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5428..5532
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5533..5601,5602..6393)
                     /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(5751..5770)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
       61 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
      121 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
      181 taccgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
      241 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
      301 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
      361 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
      421 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
      481 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
      541 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
      601 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
      661 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
      721 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
      781 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
      841 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
      901 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
      961 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     1021 cagctatgac catgattacg ccaagctcta gctagaggtc gacggtatac agacatgata
     1081 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     1141 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     1201 ggggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc
     1261 ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg
     1321 tgatggcagg ttgggcgtcg cttggtcggt catttcgaac cccagagtcc cgctcagaag
     1381 aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa
     1441 agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc
     1501 aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa
     1561 aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga
     1621 tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc
     1681 tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct
     1741 cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc
     1801 agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac
     1861 aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca
     1921 acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc
     1981 tcgtcctgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc
     2041 ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag
     2101 tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt
     2161 tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag atccgaaaat ggatatacaa
     2221 gctcccggga gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg
     2281 aatagctcag aggcagaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca
     2341 tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc
     2401 gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
     2461 cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg
     2521 cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca cagaattaat
     2581 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     2641 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     2701 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     2761 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     2821 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     2881 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     2941 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     3001 gcgcgtgggg atacccccta gagccccagc tggttctttc cgcctcagaa gccatagagc
     3061 ccaccgcatc cccagcatgc ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc
     3121 ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat
     3181 tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg
     3241 ggcaaacaac agatggctgg caactagaag gcacagtcga ggctgatcag cgggtttaaa
     3301 cgggccctct agactcgagt taggcagggg ccacctgaga agtctcggtc ttagacacgg
     3361 tggtgctggc ctcatcgaat tcaccggtgc gtccaccggt atcgatcagt gaaaagtacc
     3421 cataaataag agggtcaaaa cagggattca ggaaagcgaa gaggaagaag aagtggttga
     3481 ccggatcact cagtcggttc agcatctccg ggtcaaacca ataccaaatc cccagaacat
     3541 agtaaggagt ccagcacacg gtgaaggacg ttgcgaaggc gaccgtcatt ttaagtgtct
     3601 tcagccgtgc tctagggata ttattctttg actgattcag ctgaagttca tgggggtcct
     3661 ggtgaaggac tcttgtcagc gtaaaaatta tcttggcgtt acagatcaac atgatgaaca
     3721 gtggaataat gaaaagacaa gagaaggtga agaagttata aaacgcttgg tgccaccact
     3781 gactgaagct acagtgagtg acgcattggg agaacacttt tgtttgacca gaagagtccg
     3841 caaggtggat cattcggaag atatacagtt gtggccctgc gaagactgaa gacaggatcc
     3901 aagcgagccc caccatgctt tggcccactt tactgttgga tttaagcgcc agaggtcttg
     3961 taatggccag gctcctgtcc agggagatga ccaccatcat gaaggctggt gcgtacattg
     4021 agaacagctt gaggtaggag aggaccttgc acagcagctc tccggcatac cactgaactg
     4081 ttatattcca catcccgtcc aagggcatca caatgagggt ctccagcaga ttggccagag
     4141 tcaggtgctt cagcaacagc ttcatccggg agagtttctt ccctttctcc ttcttttgtg
     4201 tccatttctg caatttgagg agaaaggagg cattaaatgt agcgctcagc aggaagagga
     4261 agaaagtcac agtgacccgg atctttccac taagtgtaag tgtgggaaga ttcccctgca
     4321 taaggggaat gctgttgttg atggcactac agtgattctg gttctgctct ggagatgcag
     4381 agttggccat atcgatgctg gcgtcatcat cgtccttgta gtcggcgaat accaggcaga
     4441 agatgtagct cagggcgatg atcgtcttca tggtggcgct aagcttaagt ttaaacgcta
     4501 gccagcttgg gtctccctat agtgagtcgt attaatttcg ataagccagt aagcagtggg
     4561 ttctctagtt agccagagag ctctgcttat atagacctcc caccgtacac gcctaccgcc
     4621 catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg
     4681 ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa
     4741 ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac
     4801 taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg
     4861 ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac
     4921 ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa
     4981 agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc
     5041 gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata
     5101 tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg
     5161 tcaacgcgta tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag
     5221 attcttcatg caaatgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca
     5281 ctactcagcg acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc
     5341 agagcagatt gtactgagag tgcaccatag gggatcggga gatctcccga tccgtcgacg
     5401 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5461 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5521 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5581 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5641 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5701 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5761 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5821 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5881 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5941 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6001 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6061 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6121 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6181 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6241 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6301 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6361 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6421 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6481 gataatctca tgaccaaaat cccttaacgt gagttttcgt tcca
//
