LOCUS       sequence-430849-        6872 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 6872)
  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 6872)
  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: HCTR2-DuET
FEATURES             Location/Qualifiers
     source          1..6872
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    121..142
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(157..162,163..180,181..187)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    195..211
                     /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)."
     primer_bind     200..222
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     219..235
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     219..235
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    284..405
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(295..314)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     349..368
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(579..1373)
                     /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(690..709)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1300..1319
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1440..1769)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1454..1589
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1508..1527)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1754..1774
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1783..2211)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1893..1914)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2105..2124
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2257..2481
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2470..2487
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4068..4087)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4069..4087)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4132..4335)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4161..4181)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4336..4715
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4887..4906
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4981..5085
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5086..5154,5155..5946)
                     /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     complement(5304..5323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6117..6705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6606..6625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(6859..6872,1..4)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
       61 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
      121 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
      181 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      241 ttacgccaag ctctagctag aggtcgacgg tatacagaca tgataagata cattgatgag
      301 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
      361 gctattgctt tatttgtaac cattataagc tgcaataaac aagttggggt gggcgaagaa
      421 ctccagcatg agatccccgc gctggaggat catccagccg gcgtcccgga aaacgattcc
      481 gaagcccaac ctttcataga aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg
      541 cgtcgcttgg tcggtcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg
      601 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg
      661 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga
      721 tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc
      781 accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc
      841 atgcgcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc
      901 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt
      961 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca
     1021 tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc
     1081 ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct
     1141 gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca
     1201 ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc
     1261 cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc
     1321 ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac
     1381 gatcctcatc ctgtctcttg atcagatccg aaaatggata tacaagctcc cgggagcttt
     1441 ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact tctggaatag ctcagaggca
     1501 gaggcggcct cggcctctgc ataaataaaa aaaattagtc agccatgggg cggagaatgg
     1561 gcggaactgg gcggagttag gggcgggatg ggcggagtta ggggcgggac tatggttgct
     1621 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     1681 cacctggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct
     1741 ggggactttc cacaccctaa ctgacacaca ttccacagaa ttaattcgcg ttaaattttt
     1801 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     1861 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     1921 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     1981 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     2041 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     2101 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     2161 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tggggatacc
     2221 ccctagagcc ccagctggtt ctttccgcct cagaagccat agagcccacc gcatccccag
     2281 catgcctgct attgtcttcc caatcctccc ccttgctgtc ctgccccacc ccacccccca
     2341 gaatagaatg acacctactc agacaatgcg atgcaatttc ctcattttat taggaaagga
     2401 cagtgggagt ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa acaacagatg
     2461 gctggcaact agaaggcaca gtcgaggctg atcagcgggt ttaaacgggc cctctagact
     2521 cgagttaggc aggggccacc tgagaagtct cggtcttaga cacggtggtg ctggcctcat
     2581 cgaattcacc ggtgcgtcca ccggtatcga tccagttctg gaggggccca gcaccgttgg
     2641 cggcgggcag ggtgctgata ctcgtgagga ccacctgctc actgagcttt gatatgttgt
     2701 cgaagttgga tatctgtgtt gtcagactct ttctactctc tgtactagtc ctgcctctgg
     2761 tcagcctgtc ttcttggcgg tggtgcactc ccagacagca gcatgagaaa gctgctttaa
     2821 actcctcgcg gaattttcca gaaaggaaat tgtagattat tggattagca gcgctgttag
     2881 catagacaag ccagtggctg aatgtgaacc aggcatacac cgtctctcta tcctcggtgt
     2941 gtgcaaacat gccgaacact ctcttcagca cgttcaagat ggagattggc agatagcaga
     3001 ttgcgaacac aagcaggact atcatcaaca ttctggctgt tttgcgccgt gcccggatct
     3061 gtttgatttc ggccgcgaca gcactcatcc gactttttgt gggttgtcct ggacctcggg
     3121 gctgggacac aggctgcaga ggtttccact ttctctgaac cacggaagag gtcccgggaa
     3181 tttgcctgca ccagagcttc ctaaaaatct gcagataggc gagcaccatc aggcacagcg
     3241 gggccatgta agtaaccagg aaaaaacaaa tatggtacat ctttggataa atttcccccc
     3301 cccacctctc atcgcatacg gtgaacagtg tggttttgtt agccagtccc ggaaaaaccg
     3361 ttgagcactc cattacaatg gcctggggaa tcataatgat acaggacaca atccatataa
     3421 tgacgatact atttctggcg cgtttggcgg tgcttttaaa catgagtggg tgacagatag
     3481 cgtaccatcg gtccagggca atacaagaca gggtaagcac actcacagaa acgcttacag
     3541 tctggagata aggaatcacc ttacagagtg actggccgaa gaaccaagtc tcagtaatat
     3601 ccacgacgag tgtggccggc aggcaggtga ttgtcaccaa tacatctgcc agggacaggt
     3661 tgacaatgaa gtagttggtg acggtccgca tgtggtggtt cttccaaaca gccacgcaga
     3721 ccaggacatt tccgatgagt gcgactacga acacgatgat atatccagcg atcaggaccc
     3781 actcgtattc tttagggtgc agatattcgc gccacaggta ccgcaagaat tcttcgtcat
     3841 catagtcagt agggttaaga aagggctcct gtgtctcgtt gagttcggag gcgctgctcc
     3901 aattcctaca aggtgggctg tcttcaagct tagttccact catatcgatg ctggcgtcat
     3961 catcgtcctt gtagtcggcg aataccaggc agaagatgta gctcagggcg atgatcgtct
     4021 tcatggtggc gctaagctta agtttaaacg ctagccagct tgggtctccc tatagtgagt
     4081 cgtattaatt tcgataagcc agtaagcagt gggttctcta gttagccaga gagctctgct
     4141 tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg cggagttgtt
     4201 acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat tgacgtcaat
     4261 ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat tgatgtactg
     4321 ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg
     4381 aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac
     4441 gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta
     4501 ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac
     4561 atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt
     4621 taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat
     4681 tgattactat taataactag tcaataatca atgtcaacgc gtatatctgg cccgtacatc
     4741 gcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaaatgt cggtcaagcc
     4801 ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca acacacaagc
     4861 agggagcaga tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     4921 taggggatcg ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg
     4981 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5041 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5101 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5161 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5221 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5281 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5341 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5401 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5461 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5521 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5581 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5641 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5701 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5761 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     5821 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     5881 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5941 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6001 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6061 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6121 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6181 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6241 agagcgcaga taccaaatac cgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6301 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6361 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6421 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6481 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6541 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6601 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6661 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6721 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6781 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     6841 agccgaacga ccgagcgcag cgagtcagtg ag
//
