LOCUS       sequence-430897-        6752 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 6752)
  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 6752)
  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: F2R-DuET
FEATURES             Location/Qualifiers
     source          1..6752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      135..723
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     624..643
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     877..894
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1011..1032
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1047..1052,1053..1070,1071..1077)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1085..1101
                     /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     1090..1112
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1109..1125
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1109..1125
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1174..1295
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1185..1204)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1239..1258
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1469..2263)
                     /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(1580..1599)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2190..2209
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2330..2659)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2344..2479
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2398..2417)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2644..2664
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2673..3101)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2783..2804)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2995..3014
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3147..3371
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3360..3377
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4838..4857)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4839..4857)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4902..5105)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4931..4951)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5106..5485
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5657..5676
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5751..5855
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5856..5924,5925..6716)
                     /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(6074..6093)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
       61 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
      121 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
      181 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
      241 aaggtaactg gcttcagcag agcgcagata ccaaataccg ttcttctagt gtagccgtag
      301 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
      361 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
      421 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
      481 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
      541 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
      601 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
      661 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
      721 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
      781 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
      841 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      901 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
      961 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     1021 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     1081 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     1141 ctctagctag aggtcgacgg tatacagaca tgataagata cattgatgag tttggacaaa
     1201 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     1261 tatttgtaac cattataagc tgcaataaac aagttggggt gggcgaagaa ctccagcatg
     1321 agatccccgc gctggaggat catccagccg gcgtcccgga aaacgattcc gaagcccaac
     1381 ctttcataga aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg
     1441 tcggtcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg
     1501 cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt
     1561 cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg
     1621 ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat
     1681 tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct
     1741 tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct
     1801 gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt
     1861 ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga
     1921 tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc
     1981 ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa
     2041 cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac
     2101 cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg
     2161 cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc
     2221 aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc
     2281 ctgtctcttg atcagatccg aaaatggata tacaagctcc cgggagcttt ttgcaaaagc
     2341 ctaggcctcc aaaaaagcct cctcactact tctggaatag ctcagaggca gaggcggcct
     2401 cggcctctgc ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg
     2461 gcggagttag gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga
     2521 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca cacctggttg
     2581 ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc
     2641 cacaccctaa ctgacacaca ttccacagaa ttaattcgcg ttaaattttt gttaaatcag
     2701 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     2761 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     2821 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     2881 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     2941 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     3001 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     3061 caccacaccc gccgcgctta atgcgccgct acagggcgcg tggggatacc ccctagagcc
     3121 ccagctggtt ctttccgcct cagaagccat agagcccacc gcatccccag catgcctgct
     3181 attgtcttcc caatcctccc ccttgctgtc ctgccccacc ccacccccca gaatagaatg
     3241 acacctactc agacaatgcg atgcaatttc ctcattttat taggaaagga cagtgggagt
     3301 ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact
     3361 agaaggcaca gtcgaggctg atcagcgggt ttaaacgggc cctctagatt aggcaggggc
     3421 cacctgagaa gtctcggtct tagacacggt ggtgctggcc tcatcgaatt caccggtgcg
     3481 tccaccggta tcgattgtca aaagcttttt ataaatagaa ttattaagat ttgaagaaca
     3541 ggtgtccatt ttggaagcca tcagctgtcc agaagaattg taactggagg ggtcagatga
     3601 ctctttacaa cacaagatgc tgtaaacata tctctgacat tcgctagagg cgtaataata
     3661 gatcagtggg tcgatacaac aggagataga agaaacgcac acgcacagca ggtaggcgaa
     3721 ataggcagcc tcggtggtac ttgtatgact aaggaaggag taatgggcga tgaggagcac
     3781 atttgtcgga ccaaaacaaa tgatgaaaat acagaagacc gctgcagaca aaaacagagc
     3841 tcgactcttt ttactgcggt tcgccacggc actactgcta agacaccgaa taatgctgac
     3901 gtaacagact gttgagatga tcaaggggac aaagaaaaaa acagcgctga acgcggagaa
     3961 atagtacgcg taatagcctt cgagaagggt ttcgttgagc acatcgtgac aggtggttat
     4021 gttcagcccg ggaacctgga tagtctgctc cttcagcagc aggggcacca cgccggcgat
     4081 ggcaagcgcc catattgcca ggcatgtaaa agatgcgcga cccagtgtgc gccaggacag
     4141 tgactgcatt ggatatacga ctgcgaggaa tcggtcgatg ctaatcacgg tcatcaggag
     4201 gatggaggcg tacatgttgc aataaaaggc ggcagtaacg aagcggcaca gctcgcttcc
     4261 gaactgccaa tcgcttccgg aaaagtagta gcttatctta aagggcagca cgctcacaaa
     4321 gagcacgtcg gccgttgcca ggtgaagcat atatacgaca gcaggttttt tcaccttcat
     4381 cttcagaatg aacacgacga tggccatgat gttcagtggc agggaaacca caaagacccc
     4441 tgtataaacg ctcgggacaa acagagtcaa ccaactagat gtaagatatc cactggcgtc
     4501 ctcactaatg aaagcgggca actgcttctg gagcggggag ctcttattta tagataccag
     4561 gcgatattcg gtaagcccac tctcgttctt ctcttcatct tcccaaaacg gctcgtattt
     4621 atcgtttgga ttcctgagga gaaatgacct tggatccagg gtggcattcg tggctttaga
     4681 ctccggcctg cgagcgcggg tccgggcatc gatgctggcg tcatcatcgt ccttgtagtc
     4741 ggcgaatacc aggcagaaga tgtagctcag ggcgatgatc gtcttcatgg tggcgcggcc
     4801 gctaagctta agtttaaacg ctagccagct tgggtctccc tatagtgagt cgtattaatt
     4861 tcgataagcc agtaagcagt gggttctcta gttagccaga gagctctgct tatatagacc
     4921 tcccaccgta cacgcctacc gcccatttgc gtcaatgggg cggagttgtt acgacatttt
     4981 ggaaagtccc gttgattttg gtgccaaaac aaactcccat tgacgtcaat ggggtggaga
     5041 cttggaaatc cccgtgagtc aaaccgctat ccacgcccat tgatgtactg ccaaaaccgc
     5101 atcaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg aaagtcccat
     5161 aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg
     5221 ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac
     5281 tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac atacgtcatt
     5341 attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt
     5401 tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat
     5461 taataactag tcaataatca atgtcaacgc gtatatctgg cccgtacatc gcgaagcagc
     5521 gcaaaacgcc taaccctaag cagattcttc atgcaaatgt cggtcaagcc ttgccttgtt
     5581 gtagcttaaa ttttgctcgc gcactactca gcgacctcca acacacaagc agggagcaga
     5641 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca taggggatcg
     5701 ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5761 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5821 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5881 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5941 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     6001 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6061 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6121 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     6181 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6241 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6301 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6361 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6421 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6481 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6541 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6601 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6661 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6721 cagaccaagt ttactcatat atactttaga tt
//
