LOCUS       sequence-430775-        6956 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 6956)
  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 6956)
  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: CNR1-DuET
FEATURES             Location/Qualifiers
     source          1..6956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     431..450
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(669..773)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(848..867)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1039..1418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1419..1622
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1573..1593
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1667..1686
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1667..1685
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3351..3368)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3357..3581
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3627..4055
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3714..3733)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3924..3945
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4064..4084)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4069..4398
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4249..4384
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4311..4330
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4465..5259
                     /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(4519..4538)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5129..5148
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5433..5554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5470..5489)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5524..5543
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5603..5619)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5603..5619)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5616..5638)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5627..5643
                     /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)."
     promoter        complement(join(5651..5657,5658..5675,5676..5681))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5696..5717
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5834..5851)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6005..6593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6085..6104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6764..6956,1..599,600..668))
                     /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 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
       61 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
      121 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
      181 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
      241 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
      301 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
      361 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
      421 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
      481 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      541 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      601 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      661 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      721 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      781 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
      841 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
      901 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
      961 ccgacatttg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     1021 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     1081 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     1141 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     1201 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     1261 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     1321 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     1381 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     1441 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     1501 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     1561 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     1621 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact
     1681 atagggagac ccaagctggc tagcgtttaa acttaagctt agcgccacca tgaagacgat
     1741 catcgccctg agctacatct tctgcctggt attcgccgac tacaaggacg atgatgacgc
     1801 cagcatcgat atgaagagca ttttggatgg gctcgctgat accacattcc ggacaattac
     1861 aactgatctg ctgtacgtgg gttcaaacga tattcagtat gaggacataa aaggcgatat
     1921 ggcctctaaa ctgggctatt tcccacagaa gttccctctc accagcttcc gcggtagccc
     1981 ttttcaggag aaaatgactg cgggagacaa tcctcagctc gtccccgcag accaggtgaa
     2041 cattactgaa ttttacaaca agagtttgag cagctttaag gagaacgagg agaacataca
     2101 gtgcggagaa aactttatgg acattgagtg ctttatggtg ctgaatccca gtcagcagct
     2161 ggctatagcg gtgctgtccc tgacccttgg gactttcacg gtcttggaga atctgctggt
     2221 gctgtgcgtc atcttgcaca gccggtcact gcggtgtcgg ccaagttatc actttattgg
     2281 ctcactcgct gtcgctgatc tcctgggctc cgtgattttc gtttattcat tcatcgactt
     2341 ccatgtcttc caccggaaag actcacggaa cgtttttctt ttcaagcttg gaggcgttac
     2401 ggccagtttt actgctagcg taggaagcct gttcctgact gcgatcgacc gatacatcag
     2461 catccataga ccactcgctt ataagcggat tgtaacacgc ccaaaggccg tggtcgcctt
     2521 ttgcttgatg tggacgattg ccatcgtcat cgccgtgctg ccactgctcg ggtggaactg
     2581 cgaaaagttg cagtccgtgt gcagtgacat cttcccccat atcgacgaga cctacctcat
     2641 gttctggatt ggcgtaacaa gcgttctgtt gctgtttatt gtctacgctt acatgtacat
     2701 tctgtggaag gcccatagtc acgcggtccg catgatccag cgcggtaccc agaagtcaat
     2761 aattattcac acatctgaag acggcaaagt acaggtcaca agaccggacc aggctcggat
     2821 ggacatcagg cttgctaaga cactggtgtt gatactggtg gtgctgatta tctgctgggg
     2881 acctctcctc gctattatgg tgtacgacgt gttcggcaag atgaataagt tgatcaagac
     2941 cgtatttgcc ttctgttcta tgctgtgcct gcttaatagt actgtgaacc ccatcatcta
     3001 cgccctgcgc tctaaagact tgcgccacgc cttccggtcc atgtttccct cctgtgaggg
     3061 taccgcacag cccctcgaca actccatggg ggatagcgat tgccttcaca agcatgcaaa
     3121 caacgcagca tccgtccaca gggcggccga gagttgcatc aagtctaccg ttaaaatcgc
     3181 taaagttact atgtcagtca gcactgacac atctgccgaa gctctgatcg ataccggtgg
     3241 acgcaccggt gaattcgatg aggccagcac caccgtgtct aagaccgaga cttctcaggt
     3301 ggcccctgcc taactcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     3361 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3421 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3481 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3541 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3601 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3661 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3721 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3781 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3841 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3901 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3961 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4021 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     4081 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4141 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4201 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4261 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4321 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4381 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     4441 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     4501 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4561 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4621 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4681 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4741 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4801 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4861 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4921 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4981 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     5041 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     5101 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     5161 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     5221 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     5281 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     5341 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5401 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     5461 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5521 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     5581 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5641 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5701 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5761 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5821 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5881 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5941 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6001 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6061 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6121 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6181 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6241 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6301 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6361 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6421 ggcctaacta cggctacact agaagaacgg tatttggtat ctgcgctctg ctgaagccag
     6481 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6541 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6601 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6661 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6721 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6781 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6841 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6901 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccgga
//
