LOCUS       sequence-430870-        6569 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 6569)
  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 6569)
  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: MRGPRF-DuET
FEATURES             Location/Qualifiers
     source          1..6569
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      655..1243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1144..1163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1397..1414
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1531..1552
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1567..1572,1573..1590,1591..1597)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1605..1621
                     /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     1610..1632
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1629..1645
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1629..1645
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1694..1815
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1705..1724)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1759..1778
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1989..2783)
                     /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(2100..2119)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2710..2729
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2850..3179)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2864..2999
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2918..2937)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3164..3184
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3193..3621)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3303..3324)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3515..3534
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3667..3891
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3880..3897
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5175..5194)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5176..5194)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5239..5442)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5268..5288)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5443..5822
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5994..6013
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6088..6192
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6193..6261,6262..6569,1..484)
                     /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(6411..6430)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
       61 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
      121 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
      181 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
      241 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
      301 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
      361 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
      421 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
      481 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
      541 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
      601 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
      661 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
      721 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
      781 agcgcagata ccaaataccg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
      841 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
      901 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
      961 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     1021 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     1081 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     1141 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     1201 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     1261 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     1321 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     1381 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     1441 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     1501 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     1561 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     1621 atttcacaca ggaaacagct atgaccatga ttacgccaag ctctagctag aggtcgacgg
     1681 tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     1741 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     1801 tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat
     1861 catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt
     1921 ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt cgaaccccag
     1981 agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga
     2041 gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca
     2101 atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag
     2161 tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca
     2221 tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg
     2281 gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc
     2341 atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc
     2401 ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga
     2461 gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt
     2521 cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac
     2581 gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc ggtcttgaca
     2641 aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt
     2701 gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg
     2761 tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagatccg
     2821 aaaatggata tacaagctcc cgggagcttt ttgcaaaagc ctaggcctcc aaaaaagcct
     2881 cctcactact tctggaatag ctcagaggca gaggcggcct cggcctctgc ataaataaaa
     2941 aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag gggcgggatg
     3001 ggcggagtta ggggcgggac tatggttgct gactaattga gatgcatgct ttgcatactt
     3061 ctgcctgctg gggagcctgg ggactttcca cacctggttg ctgactaatt gagatgcatg
     3121 ctttgcatac ttctgcctgc tggggagcct ggggactttc cacaccctaa ctgacacaca
     3181 ttccacagaa ttaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3241 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     3301 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     3361 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     3421 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     3481 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     3541 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     3601 atgcgccgct acagggcgcg tggggatacc ccctagagcc ccagctggtt ctttccgcct
     3661 cagaagccat agagcccacc gcatccccag catgcctgct attgtcttcc caatcctccc
     3721 ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc agacaatgcg
     3781 atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc agggtcaagg
     3841 aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca gtcgaggctg
     3901 atcagcgggt ttaaacgggc cctctagact cgagttaggc aggggccacc tgagaagtct
     3961 cggtcttaga cacggtggtg ctggcctcat cgaattcacc ggtgcgtcca ccggtatcga
     4021 tagaggcgtt gcccgggggg cattgcatct ccatagtgac tgtattaggg gtagagccgc
     4081 cagcttcccc gagctcagcc ccgtcccgaa gtgctcgctg aaatactacc cggagaggct
     4141 cccacaggcg ctgggacttg tcgcgtcctg cgaggaagta aacgattggt ttggcactgc
     4201 tattaataca gatacacagg tctgtcacgt actcaggaaa gggggcggga atctggaaaa
     4261 cccagaacag aaaccagtcg atgcccagat aaatagatga caccaaaaaa acagacacca
     4321 tggcgaggat cacgtgattg agctttgcag acctctgcct gcgccgcgct ctacactcca
     4381 catgaaggat cagagccaga catgggagaa ccatcagggg gcagcagagc aggaacagca
     4441 aaattccgag gaatatatcc atatgcctgc atgcggcgcc gggagcgcct ctgcccagga
     4501 acacacaaaa gtagttatgc agacaggtca ccaggaggct gagcacccaa agaagcgcac
     4561 acaccacagc actcagcctc ttaggtcttc ggcgccagta ccaggccgga aaaattacgg
     4621 aagcacacct ctcggcagaa actgcgggca gcagtgaaac cccagtcagg aacatgcaaa
     4681 gaccgagcac cctacacacg cttcttatat aatctgcgaa ggtcccgagg aatcctccgg
     4741 tgttgagaat tgagaacacg gctttactaa aaaggtatcc tacatctgca gatgcaaggt
     4801 gcaggaaata aatagaaaat ggatttcttt ttatggagaa accaaagaac cacagcacga
     4861 ggccgttacc tacaagtccg caaaggcaga ggagaaggaa aatgtaattc atgacggctg
     4921 gaggggggag catagctatc tgctcaattg tgaggaaacc gcgggagtac agttcggggg
     4981 cttctgagag cccggggcac attttgttcc ggttcccagg gtgcgcctcc catgagcagt
     5041 tgcctgccat atcgatgctg gcgtcatcat cgtccttgta gtcggcgaat accaggcaga
     5101 agatgtagct cagggcgatg atcgtcttca tggtggcgct aagcttaagt ttaaacgcta
     5161 gccagcttgg gtctccctat agtgagtcgt attaatttcg ataagccagt aagcagtggg
     5221 ttctctagtt agccagagag ctctgcttat atagacctcc caccgtacac gcctaccgcc
     5281 catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg
     5341 ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa
     5401 ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac
     5461 taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg
     5521 ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac
     5581 ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa
     5641 agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc
     5701 gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata
     5761 tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg
     5821 tcaacgcgta tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag
     5881 attcttcatg caaatgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca
     5941 ctactcagcg acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc
     6001 agagcagatt gtactgagag tgcaccatag gggatcggga gatctcccga tccgtcgacg
     6061 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     6121 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     6181 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     6241 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     6301 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     6361 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     6421 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     6481 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     6541 gtaagagaat tatgcagtgc tgccataac
//
