LOCUS       sequence-430729-        6746 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 6746)
  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 6746)
  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: PTGER1-DuET
FEATURES             Location/Qualifiers
     source          1..6746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(55..72)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    61..285
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      331..759
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(418..437)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     628..649
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(768..788)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        773..1102
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      953..1088
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1015..1034
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1169..1963
                     /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(1223..1242)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1833..1852
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2137..2258
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2174..2193)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2228..2247
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2307..2323)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2307..2323)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2320..2342)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2331..2347
                     /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(2355..2361,2362..2379,2380..2385))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2400..2421
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2538..2555)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2709..3297)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2789..2808)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3468..4259,4260..4328))
                     /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     4091..4110
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4329..4433)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4508..4527)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4699..5078
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5079..5282
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5233..5253
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5327..5346
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5327..5345
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 aggtggcccc tgcctaactc gagtctagag ggcccgttta aacccgctga tcagcctcga
       61 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
      121 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
      181 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
      241 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
      301 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
      361 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
      421 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      481 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      541 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
      601 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
      661 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
      721 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
      781 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
      841 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
      901 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
      961 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     1021 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     1081 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     1141 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     1201 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     1261 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     1321 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     1381 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     1441 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     1501 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     1561 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     1621 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     1681 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     1741 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     1801 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     1861 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     1921 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     1981 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     2041 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     2101 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     2161 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     2221 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     2281 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2341 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     2401 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2461 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2521 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2581 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2641 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2701 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2761 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2821 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2881 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2941 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3001 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3061 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3121 tggtggccta actacggcta cactagaaga acggtatttg gtatctgcgc tctgctgaag
     3181 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3241 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3301 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3361 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3421 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3481 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3541 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3601 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3661 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3721 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3781 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3841 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3901 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3961 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4021 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4081 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4141 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4201 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4261 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4321 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4381 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4441 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     4501 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     4561 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
     4621 ccgacatttg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     4681 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     4741 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     4801 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     4861 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     4921 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     4981 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     5041 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     5101 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     5161 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     5221 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     5281 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact
     5341 atagggagac ccaagctggc tagcgtttaa acttaagctt agcgccacca tgaagacgat
     5401 catcgccctg agctacatct tctgcctggt attcgccgac tacaaggacg atgatgacgc
     5461 cagcatcgat atgagtccat gtggtccact gaacctgagc ctcgccggag aggcgacgac
     5521 ctgtgcagcc ccatgggtcc ccaacactag cgcggtgccc ccaagtggag cctcaccagc
     5581 cctccctatc tttagcatga cgctgggagc tgtgagtaat ctgctggccc ttgctctgct
     5641 cgcccaagct gctggccgcc tcagacgccg gcgaagcgcc gccacgtttc ttctcttcgt
     5701 ggcaagcctg ctggcaactg accttgccgg ccatgtaatc cccggcgctt tggtcctgag
     5761 gctgtatact gccggaagag cgccggctgg cggcgcatgc cactttctcg gcggatgtat
     5821 ggtctttttc ggtctttgcc ccctgctttt gggatgcggt atggcggtgg aacgatgcgt
     5881 cggggtgacc agacctctgc tccacgcagc ccgcgtttct gtggcacgcg ccagattggc
     5941 tttggctgca gtcgcagccg tggcccttgc agtagccctt cttcctcttg cacgggtggg
     6001 gcgatatgaa ctgcaatatc ctgggacttg gtgcttcatc gggctgggac cacccggagg
     6061 atggagacaa gcactgctcg ccgggctttt tgccagcctg ggcctcgtgg cgttgctggc
     6121 agctctggtt tgcaatacgc tgtccggact cgccctcctg cgcgcaaggt ggagacgaag
     6181 gtcaaggcga ccaccacccg ctagtggacc cgatagtagg aggaggtggg gagcgcatgg
     6241 acctaggagt gcgtccgcct ccagcgcgag tagcattgcc tcagccagta cttttttcgg
     6301 cggctcccgg tctagcggct ctgcaagacg agctcgcgca cacgatgtgg aaatggtagg
     6361 tcagctcgta ggtattatgg tggttagctg catttgctgg agccccatgc ttgtcctggt
     6421 tgccctcgca gtggggggct ggagctcaac gtcactccag cgccccctgt tcctggcggt
     6481 cagacttgcc tcttggaacc agatcctgga tccctgggtc tacatcctgc tgagacaggc
     6541 tgtcttgcgc caactcctga ggctccttcc cccccgcgca ggggcgaagg gaggtccagc
     6601 tggcctgggg ctgactcctt cagcttggga ggcttcctcc ctgaggtcat cacgacactc
     6661 cgggctgtcc cacttcatcg ataccggtgg acgcaccggt gaattcgatg aggccagcac
     6721 caccgtgtct aagaccgaga cttctc
//
