LOCUS       sequence-430891-        6704 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 6704)
  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 6704)
  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: SSTR4-DuET
FEATURES             Location/Qualifiers
     source          1..6704
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(204..308)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(383..402)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        574..953
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        954..1157
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1108..1128
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1202..1221
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1202..1220
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2634..2651)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2640..2864
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2910..3338
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2997..3016)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3207..3228
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3347..3367)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3352..3681
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3532..3667
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3594..3613
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3748..4542
                     /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(3802..3821)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4412..4431
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4716..4837
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4753..4772)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4807..4826
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4886..4902)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4886..4902)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4899..4921)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4910..4926
                     /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(4934..4940,4941..4958,4959..4964))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4979..5000
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5117..5134)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5288..5876)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5368..5387)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6047..6704,1..134,135..203))
                     /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     6670..6689
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
       61 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
      121 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
      181 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
      241 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
      301 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
      361 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
      421 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
      481 caaggcaagg cttgaccgac atttgcatga agaatctgct tagggttagg cgttttgcgc
      541 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
      601 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      661 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      721 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      781 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      841 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      901 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      961 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     1021 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
     1081 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
     1141 tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa
     1201 ttaatacgac tcactatagg gagacccaag ctggctagcg tttaaactta agcttagcgg
     1261 ccgcgccacc atgaagacga tcatcgccct gagctacatc ttctgcctgg tattcgccga
     1321 ctacaaggac gatgatgacg ccagcatcga tatgtcagct ccttccacct tgcctcctgg
     1381 gggcgaagaa ggcctgggca ccgcttggcc cagtgcagca aacgcttctt ctgcccccgc
     1441 agaagcggaa gaggccgttg caggaccagg ggatgccaga gctgctggca tggttgcaat
     1501 ccagtgcatt tacgccctgg tgtgtctggt cggactcgtc ggcaatgcat tggttatctt
     1561 cgtgattttg aggtacgcta aaatgaagac agcgacaaat atatatcttt tgaatctggc
     1621 tgtggccgac gagctgttca tgctgagtgt gccatttgtc gcctcatcag ccgctctccg
     1681 acactggccc ttcgggagcg tgctgtgccg agccgtgctg tccgtggacg ggctcaatat
     1741 gttcaccagc gttttttgcc tgaccgtgtt gagcgtcgac agatacgttg ccgtggtgca
     1801 tcctttgcgg gcggctacat acaggcgccc atcagttgcc aagctgatta atctgggcgt
     1861 gtggttggcg agtctgctgg ttacactgcc aatcgctatc tttgcggata cccgacctgc
     1921 ccgcggcggt caggccgtag cctgcaatct ccagtggcca catcctgctt ggtctgctgt
     1981 gttcgtcgtc tacacctttc ttctcggttt cctgttgccc gtgctggcca tcggcttgtg
     2041 ctacctcttg atagtgggca agatgagggc ggtggctctg cgagccggtt ggcagcagcg
     2101 acgacgcagc gagaagaaga taactcgcct tgttttgatg gtagtcgttg tgttcgtact
     2161 ttgctggatg cccttctatg tcgtgcagct gctcaacttg tttgtgacta gcctcgacgc
     2221 caccgtgaac cacgtctctc tgatcctcag ttacgcaaat agctgcgcca acccaatact
     2281 gtacgggttc ctctctgata atttccgcag atttttccag agagtgttgt gtctgcgatg
     2341 ttgcctgctg gagggagcgg ggggagccga agaagagccg ctcgactatt acgcaactgc
     2401 cctgaagagc aagggaggcg cgggctgtat gtgccctcca ctgccctgtc agcaagaagc
     2461 gcttcagccc gaaccaggaa gaaagcgcat tccccttacc agaaccacaa cctttatcga
     2521 taccggtgga cgcaccggtg aattcgatga ggccagcacc accgtgtcta agaccgagac
     2581 ttctcaggtg gcccctgcct aatctagagg gcccgtttaa acccgctgat cagcctcgac
     2641 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2701 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2761 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2821 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2881 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2941 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3001 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3061 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3121 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3181 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3241 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3301 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3361 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3421 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3481 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3541 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3601 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     3661 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     3721 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     3781 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     3841 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     3901 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     3961 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4021 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4081 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4141 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4201 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4261 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4321 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4381 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4441 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4501 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     4561 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     4621 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4681 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     4741 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4801 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     4861 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4921 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4981 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5041 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5101 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5161 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5221 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5281 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5341 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5401 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5461 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5521 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5581 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5641 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5701 ggtggcctaa ctacggctac actagaagaa cggtatttgg tatctgcgct ctgctgaagc
     5761 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5821 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5881 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5941 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6001 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6061 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6121 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6181 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6241 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6301 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6361 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6421 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6481 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6541 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6601 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6661 caatacggga taataccgcg ccacatagca gaactttaaa agtg
//
