LOCUS       sequence-430732-        6713 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 6713)
  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 6713)
  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: SSTR1-DuET
FEATURES             Location/Qualifiers
     source          1..6713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..56)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    45..269
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      315..743
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(402..421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     612..633
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(752..772)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        757..1086
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      937..1072
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     999..1018
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1153..1947
                     /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(1207..1226)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1817..1836
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2121..2242
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2158..2177)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2212..2231
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2291..2307)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2291..2307)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2304..2326)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2315..2331
                     /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(2339..2345,2346..2363,2364..2369))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2384..2405
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2522..2539)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2693..3281)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2773..2792)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3452..4243,4244..4312))
                     /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     4075..4094
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4313..4417)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4492..4511)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4683..5062
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5063..5266
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5217..5237
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5311..5330
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5311..5329
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 actcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
       61 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
      121 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
      181 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
      241 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
      301 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
      361 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
      421 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
      481 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
      541 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
      601 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
      661 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
      721 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
      781 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
      841 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
      901 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
      961 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     1021 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     1081 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     1141 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     1201 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     1261 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     1321 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     1381 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     1441 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     1501 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     1561 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     1621 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     1681 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     1741 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     1801 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     1861 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     1921 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     1981 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     2041 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     2101 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     2161 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     2221 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     2281 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     2341 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     2401 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     2461 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     2521 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     2581 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2641 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2701 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2761 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2821 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2881 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2941 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3001 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3061 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3121 gctacactag aagaacggta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3181 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3241 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3301 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3361 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3421 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3481 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3541 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3601 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3661 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3721 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3781 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3841 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3901 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3961 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4021 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4081 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4141 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4201 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4261 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4321 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4381 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4441 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     4501 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     4561 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca tttgcatgaa
     4621 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
     4681 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     4741 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     4801 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     4861 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     4921 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     4981 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     5041 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     5101 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     5161 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     5221 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
     5281 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
     5341 tggctagcgt ttaaacttaa gcttagcgcc accatgaaga cgatcatcgc cctgagctac
     5401 atcttctgcc tggtattcgc cgactacaag gacgatgatg acgccagcat cgatatgttt
     5461 cccaacggca cggcatcatc accaagttct tccccaagcc ctagccccgg atcatgcggg
     5521 gagggtggtg ggtcccgcgg acccggagca ggcgccgctg atggaatgga ggagccagga
     5581 cgcaatgcta gtcagaacgg cacactcagc gaggggcagg gctctgctat cctcatcagc
     5641 tttatctact ccgtggtctg cctggtcggg ctgtgcggaa attccatggt catctacgtt
     5701 attctgcgct acgcaaaaat gaaaactgca acaaatattt acatcctgaa cttggccatc
     5761 gcagatgaac tgctgatgct gtccgttccc tttctggtta ccagcactct gctgcgccat
     5821 tggccattcg gcgcgctgct ctgtagactg gtcctgagtg tggacgccgt gaatatgttc
     5881 acaagcattt actgccttac cgtgctctcc gttgacaggt acgtggccgt cgtgcaccca
     5941 atcaaggcag ccagatatag acggcctaca gtggccaaag tagttaacct cggagtgtgg
     6001 gttttgtctc tgctggttat tctgccaatt gtggtgttct cccgcacagc tgctaattcc
     6061 gacggcaccg tagcatgcaa tatgctgatg ccggaacccg ctcagcggtg gctcgtgggg
     6121 tttgttttgt acacgttcct catggggttc cttctccctg tgggtgccat atgtctgtgt
     6181 tacgtcctta tcatagctaa gatgcgcatg gttgcactta aggcaggctg gcaacagagg
     6241 aaaaggtccg agcgcaagat cacgctgatg gtcatgatgg tagtgatggt ttttgtcatt
     6301 tgttggatgc cattttacgt cgtgcagttg gtgaacgtct tcgcagaaca ggacgacgcc
     6361 acagtctccc agctttctgt catccttgga tacgccaata gttgtgcaaa cccgatcctg
     6421 tatggattct tgtccgacaa tttcaagaga tcttttcaga gaattctgtg tctgtcttgg
     6481 atggacaacg ccgctgagga accagtggac tactacgcaa ccgcgcttaa gagccgagca
     6541 tattctgtgg aggatttcca gccagagaat ctggagagcg gcggggtgtt caggaatggc
     6601 acttgtacta gccgaatcac caccctgatc gataccggtg gacgcaccgg tgaattcgat
     6661 gaggccagca ccaccgtgtc taagaccgag acttctcagg tggcccctgc cta
//
