LOCUS       sequence-430734-        6794 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 6794)
  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 6794)
  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: SSTR3-DuET
FEATURES             Location/Qualifiers
     source          1..6794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(79..96)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    85..309
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      355..783
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(442..461)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     652..673
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(792..812)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        797..1126
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      977..1112
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1039..1058
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1193..1987
                     /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(1247..1266)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1857..1876
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2161..2282
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2198..2217)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2252..2271
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2331..2347)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2331..2347)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2344..2366)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2355..2371
                     /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(2379..2385,2386..2403,2404..2409))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2424..2445
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2562..2579)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2733..3321)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2813..2832)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3492..4283,4284..4352))
                     /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     4115..4134
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4353..4457)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4532..4551)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4723..5102
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5103..5306
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5257..5277
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5351..5370
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5351..5369
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ccgtgtctaa gaccgagact tctcaggtgg cccctgccta actcgagtct agagggcccg
       61 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
      121 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
      181 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
      241 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
      301 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
      361 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      421 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      481 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      541 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
      601 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      661 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
      721 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
      781 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
      841 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
      901 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
      961 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     1021 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     1081 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     1141 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     1201 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     1261 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     1321 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     1381 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     1441 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     1501 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     1561 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     1621 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     1681 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     1741 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     1801 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     1861 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     1921 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     1981 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     2041 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     2101 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     2161 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     2221 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     2281 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     2341 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     2401 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     2461 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     2521 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     2581 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     2641 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2701 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2761 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2821 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2881 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2941 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3001 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3061 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3121 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacggta
     3181 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3241 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3301 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3361 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3421 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3481 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     3541 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     3601 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     3661 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3721 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3781 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3841 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3901 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3961 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4021 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4081 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4141 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4201 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4261 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4321 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4381 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4441 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     4501 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4561 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     4621 aagctacaac aaggcaaggc ttgaccgaca tttgcatgaa gaatctgctt agggttaggc
     4681 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     4741 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     4801 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     4861 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     4921 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     4981 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     5041 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     5101 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     5161 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     5221 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     5281 ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc
     5341 ttatcgaaat taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa
     5401 gcttagcgcc accatgaaga cgatcatcgc cctgagctac atcttctgcc tggtattcgc
     5461 cgactacaag gacgatgatg acgccagcat cgatatggac atgctccatc cttcttcagt
     5521 ttccacaacc agcgagcccg aaaacgcatc tagcgcttgg ccccccgatg caaccttggg
     5581 caacgtgtca gctggcccat ctccggccgg attggctgtg tctggggtac tgattccgct
     5641 cgtgtacttg gtcgtgtgtg tcgtgggcct gctcggtaat tcacttgtca tttatgtggt
     5701 gcttcggcat accgcatctc cttcagtcac caatgtgtat attcttaacc ttgctctggc
     5761 ggatgagttg tttatgctcg ggctgccctt cctcgccgct cagaacgccc tgagctactg
     5821 gcctttcggg tccctcatgt gtcgcctggt gatggcagtg gatggcatca atcagtttac
     5881 cagtattttc tgcctgaccg tgatgagcgt ggaccgctat ttggccgtag tgcatccaac
     5941 cagaagtgct agatggcgca ccgcccctgt ggcccgcacg gtgagtgctg ccgtttgggt
     6001 tgcaagtgct gtggtagtgc tccccgtggt tgttttcagt ggagtgccac ggggtatgtc
     6061 cacctgtcat atgcagtggc cagagcctgc tgccgcttgg agagccggtt tcatcattta
     6121 caccgctgcc ctgggattct ttgggccact gctcgtcatc tgtctctgtt acctgctgat
     6181 cgtggtgaaa gtgagaagcg cgggtcggag agtgtgggcg cctagttgtc agcgacgccg
     6241 caggtctgag cgacgcgtta ctagaatggt ggtggcggta gttgcactct tcgtgttgtg
     6301 ttggatgcct ttttacgtgt tgaatattgt gaacgtcgtg tgccctctgc cagaagaacc
     6361 cgcatttttc ggcttgtatt tcctcgtcgt cgcactcccc tacgccaact catgcgcaaa
     6421 tcctatcctg tatgggttcc ttagttaccg cttcaaacag ggctttaggc gggtgctgct
     6481 ccgcccatcc cggcgcgtac gctcacagga gcccactgta ggcccacccg agaaaacaga
     6541 agaagaggac gaggaggaag aagacggcga ggaatctaga gagggcggca aaggtaaaga
     6601 gatgaacggc agagtgtcac agatcactca gcccggcacc agcggacagg aacgaccccc
     6661 cagcagagtg gcgagcaaag aacagcagct tctcccccaa gaggcctcca caggggaaaa
     6721 aagttctaca atgcgcatca gctacctgat cgataccggt ggacgcaccg gtgaattcga
     6781 tgaggccagc acca
//
