LOCUS       sequence-430795-        6695 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 6695)
  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 6695)
  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: F2RL3-DuET
FEATURES             Location/Qualifiers
     source          1..6695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(96..115)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      909..1497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1398..1417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1651..1668
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1785..1806
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1821..1826,1827..1844,1845..1851)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1859..1875
                     /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     1864..1886
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1883..1899
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1883..1899
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1948..2069
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1959..1978)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2013..2032
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2243..3037)
                     /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(2354..2373)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2964..2983
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3104..3433)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3118..3253
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3172..3191)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3418..3438
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3447..3875)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3557..3578)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3769..3788
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3921..4145
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4134..4151
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5555..5574)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5556..5574)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5619..5822)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5648..5668)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5823..6202
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6374..6393
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6468..6572
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6573..6641,6642..6695,1..738)
                     /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"
ORIGIN
        1 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
       61 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
      121 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
      181 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
      241 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
      301 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
      361 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
      421 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
      481 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
      541 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
      601 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
      661 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
      721 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
      781 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
      841 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
      901 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
      961 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     1021 actggcttca gcagagcgca gataccaaat accgttcttc tagtgtagcc gtagttaggc
     1081 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     1141 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     1201 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     1261 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     1321 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     1381 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     1441 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     1501 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     1561 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     1621 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     1681 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     1741 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     1801 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     1861 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagctctag
     1921 ctagaggtcg acggtataca gacatgataa gatacattga tgagtttgga caaaccacaa
     1981 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     2041 taaccattat aagctgcaat aaacaagttg gggtgggcga agaactccag catgagatcc
     2101 ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca
     2161 tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc
     2221 atttcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc
     2281 gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc
     2341 caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac
     2401 ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca
     2461 agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgcgc gccttgagcc
     2521 tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga
     2581 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga
     2641 atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata
     2701 ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata
     2761 gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg
     2821 tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca
     2881 ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat
     2941 cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg
     3001 ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct catcctgtct
     3061 cttgatcaga tccgaaaatg gatatacaag ctcccgggag ctttttgcaa aagcctaggc
     3121 ctccaaaaaa gcctcctcac tacttctgga atagctcaga ggcagaggcg gcctcggcct
     3181 ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa ctgggcggag
     3241 ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca
     3301 tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact
     3361 aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc
     3421 ctaactgaca cacattccac agaattaatt cgcgttaaat ttttgttaaa tcagctcatt
     3481 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     3541 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     3601 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     3661 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     3721 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     3781 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     3841 acccgccgcg cttaatgcgc cgctacaggg cgcgtgggga taccccctag agccccagct
     3901 ggttctttcc gcctcagaag ccatagagcc caccgcatcc ccagcatgcc tgctattgtc
     3961 ttcccaatcc tcccccttgc tgtcctgccc caccccaccc cccagaatag aatgacacct
     4021 actcagacaa tgcgatgcaa tttcctcatt ttattaggaa aggacagtgg gagtggcacc
     4081 ttccagggtc aaggaaggca cgggggaggg gcaaacaaca gatggctggc aactagaagg
     4141 cacagtcgag gctgatcagc gggtttaaac gggccctcta gactcgagtt aggcaggggc
     4201 cacctgagaa gtctcggtct tagacacggt ggtgctggcc tcatcgaatt caccggtgcg
     4261 tccaccggta tcgatttgca gcagtgaaga gtgggtcccc ataccccggc tgcccccctc
     4321 ggcgctggct ttggaggcaa cagtgtcccc tggacttctc tgaaagaggc cggctcggac
     4381 cttatctctg aactcggcgc ttacatagta gtaaataaaa ggatccacgc aggaattcaa
     4441 ggtgctcagg gccaagctgg gcacataggc gccatacagg ttaccccagg ctgaggggga
     4501 agggtcgcta taatgcagga gcaggaggag gttgctcgga acgaagaagg ccactgcgct
     4561 cgccagtact actgcagtca gtctaagggc gtgtccgtat cgcctacctg aggcagccaa
     4621 agtgtgcaat gtggccccat aacagagcag catcgccagg agaggcagaa agcatcccaa
     4681 cagagccaag caagtaaatg cgggctgcca gtgtgaagcc tgcgcgtcga gaggcagtgc
     4741 gtcgtggcac agcactctgt cggagcgagc caggcgaaaa gtctgtctct gcagagtgag
     4801 tgggagtgcg agggcagcgg ccatcagcca ggcagccatg cacaaaccca gtgccaggcg
     4861 cctgcctcta agagcccggg ctctcagcgg atggaccaat gccaggtatc tatccaagct
     4921 caccgcggcc agaaggagca ctgacccgta catgtgcccg tacaaagcgg cagtggcgag
     4981 cctacaggcg gcctctccga atggccaccg ctgcccccga agatggtagg caatgcgtgg
     5041 aggcagggcg agtgccaaga ggaggtcggc ggcggccagg ttcataagaa gcatcgtaga
     5101 ggggagccgt ggggcctggg tagccagaac ccacagagcc agaccgttgg cgggcaggcc
     5161 cacaacaagc accaaaccgt agagggctgg cacgagtctg gtaggcaccc accccagcag
     5221 cagtgctcta ctgctgtctg gcaactccag agtatcgcta tcatttgcac agacttgacc
     5281 gggatagccc ctcggggcgg gaagtatgct gggagtagag tcgtcaccgc ctcccgtgct
     5341 tccgctttcg tcatacacag atggagtctg tgtgcctccg ctaagggaaa aacccagcac
     5401 aagaggccac agcagaagcc gtccccacat atcgatgctg gcgtcatcat cgtccttgta
     5461 gtcggcgaat accaggcaga agatgtagct cagggcgatg atcgtcttca tggtggcgct
     5521 aagcttaagt ttaaacgcta gccagcttgg gtctccctat agtgagtcgt attaatttcg
     5581 ataagccagt aagcagtggg ttctctagtt agccagagag ctctgcttat atagacctcc
     5641 caccgtacac gcctaccgcc catttgcgtc aatggggcgg agttgttacg acattttgga
     5701 aagtcccgtt gattttggtg ccaaaacaaa ctcccattga cgtcaatggg gtggagactt
     5761 ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga tgtactgcca aaaccgcatc
     5821 accatggtaa tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag
     5881 gtcatgtact gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc
     5941 gtacttggca tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc
     6001 acccattgac gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt
     6061 gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat
     6121 gtaacgcgga actccatata tgggctatga actaatgacc ccgtaattga ttactattaa
     6181 taactagtca ataatcaatg tcaacgcgta tatctggccc gtacatcgcg aagcagcgca
     6241 aaacgcctaa ccctaagcag attcttcatg caaatgtcgg tcaagccttg ccttgttgta
     6301 gcttaaattt tgctcgcgca ctactcagcg acctccaaca cacaagcagg gagcagatac
     6361 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatag gggatcggga
     6421 gatctcccga tccgtcgacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     6481 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     6541 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     6601 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     6661 aagtaaaaga tgctgaagat cagttgggtg cacga
//
