LOCUS       sequence-430793-        6731 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 6731)
  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 6731)
  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: F2RL1-DuET
FEATURES             Location/Qualifiers
     source          1..6731
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      151..739
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     640..659
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     893..910
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1027..1048
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1063..1068,1069..1086,1087..1093)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1101..1117
                     /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     1106..1128
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1125..1141
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1125..1141
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1190..1311
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1201..1220)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1255..1274
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1485..2279)
                     /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(1596..1615)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2206..2225
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2346..2675)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2360..2495
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2414..2433)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2660..2680
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2689..3117)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2799..2820)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3011..3030
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3163..3387
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3376..3393
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4833..4852)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4834..4852)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4897..5100)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4926..4946)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5101..5480
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5652..5671
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5746..5850
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5851..5919,5920..6711)
                     /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(6069..6088)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
       61 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
      121 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
      181 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
      241 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa ataccgttct
      301 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
      361 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
      421 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
      481 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
      541 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
      601 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
      661 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
      721 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
      781 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
      841 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
      901 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
      961 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     1021 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     1081 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     1141 ccatgattac gccaagctct agctagaggt cgacggtata cagacatgat aagatacatt
     1201 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     1261 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt tggggtgggc
     1321 gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac
     1381 gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtgatggcag
     1441 gttgggcgtc gcttggtcgg tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca
     1501 agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg
     1561 aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg
     1621 tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca
     1681 ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg
     1741 tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct
     1801 tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg
     1861 cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc
     1921 attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc
     1981 tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc
     2041 acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc
     2101 agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct
     2161 gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg
     2221 aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg
     2281 cgaaacgatc ctcatcctgt ctcttgatca gatccgaaaa tggatataca agctcccggg
     2341 agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca
     2401 gaggcagagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga
     2461 gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg
     2521 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
     2581 tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
     2641 gagcctgggg actttccaca ccctaactga cacacattcc acagaattaa ttcgcgttaa
     2701 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     2761 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     2821 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     2881 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     2941 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     3001 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     3061 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtggg
     3121 gataccccct agagccccag ctggttcttt ccgcctcaga agccatagag cccaccgcat
     3181 ccccagcatg cctgctattg tcttcccaat cctccccctt gctgtcctgc cccaccccac
     3241 cccccagaat agaatgacac ctactcagac aatgcgatgc aatttcctca ttttattagg
     3301 aaaggacagt gggagtggca ccttccaggg tcaaggaagg cacgggggag gggcaaacaa
     3361 cagatggctg gcaactagaa ggcacagtcg aggctgatca gcgggtttaa acgggccctc
     3421 tagactcgag ttaggcaggg gccacctgag aagtctcggt cttagacacg gtggtgctgg
     3481 cctcatcgaa ttcaccggtg cgtccaccgg tatcgatgta gctagtcttc accgtagtac
     3541 tagaactgct gtaagaagaa gatttccgag agtgtttctt agaagtaagg ctgacctgca
     3601 tctgcttgac agttcgcacg ctccgacaca gaagggcgtt cttggcgtgg tcccgaaaat
     3661 cgtgtgagac aaagtaatac acgaaagggt ctatacatga attaagggtg ctcagacaaa
     3721 gtgccacaat gtacagggca taaacatggg actggccctg ggacttaatc agaaagtaat
     3781 gcaccaccaa cagaagatta gagggcgtaa agcagatgag gtacatggcc agtacagtga
     3841 cgatcaactt gatagcccgt ttccgctttt tctctgaatt ttcgtccatg gcagagcttc
     3901 taagcatgcg gatcatcagc acataggcac tagcagtcag aaatgcggga aagaggaaga
     3961 ctccaatggc caaactcagg aaataattga acatgtcgcc taccagcagc tgctcaggaa
     4021 ggacgtcgtg acaagtagtg atgttcagag cggggatgaa tatggtttgc ttaaccacat
     4081 aaagcggaat tgtcacgagc aggatcagca accatatagc caaggaaata ccgatcgcga
     4141 tgttggcctt cttccgagag tgacccatcg ggttcacgat gacccaatac ctttgcacgc
     4201 tcaggcacgt catgaagagg atagaacagt acatatttcc gtaaaagaat ccaataagga
     4261 cattgcagag tgcctctcca tagatccagt tattgccgtg gatgtggtac gctatcttca
     4321 agggaaacca aatgacacta agcagatcgg ccagggcaag attagccatg tagatgaccg
     4381 ctgggtgttt cttcttagtt ctgaacaaga aaacccagag ggccatccca ttgcttggaa
     4441 ggcccaccac gaaaacgata gtgtaaacaa ttgggaggaa aactgtcgtc agcttaccag
     4501 tcaagacgga tgcactgaat tcgtcaactg aaaacacagt ttcgacggtc actcccttac
     4561 ctgtcacgtg gctggtgcca tctaccttgc cgatcagtga tcgtcccttg gatgacctgt
     4621 tggtgccctg aattgtgcca ctgcaggaga gagatgcagc gagcaggatt gccgccccga
     4681 gcagccacgc ggcagaggga ctccgcatat cgatgctggc gtcatcatcg tccttgtagt
     4741 cggcgaatac caggcagaag atgtagctca gggcgatgat cgtcttcatg gtggcgctaa
     4801 gcttaagttt aaacgctagc cagcttgggt ctccctatag tgagtcgtat taatttcgat
     4861 aagccagtaa gcagtgggtt ctctagttag ccagagagct ctgcttatat agacctccca
     4921 ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag ttgttacgac attttggaaa
     4981 gtcccgttga ttttggtgcc aaaacaaact cccattgacg tcaatggggt ggagacttgg
     5041 aaatccccgt gagtcaaacc gctatccacg cccattgatg tactgccaaa accgcatcac
     5101 catggtaata gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt
     5161 catgtactgg gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt
     5221 acttggcata tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac
     5281 ccattgacgt caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga
     5341 cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt
     5401 aacgcggaac tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata
     5461 actagtcaat aatcaatgtc aacgcgtata tctggcccgt acatcgcgaa gcagcgcaaa
     5521 acgcctaacc ctaagcagat tcttcatgca aatgtcggtc aagccttgcc ttgttgtagc
     5581 ttaaattttg ctcgcgcact actcagcgac ctccaacaca caagcaggga gcagatactg
     5641 gcttaactat gcggcatcag agcagattgt actgagagtg caccataggg gatcgggaga
     5701 tctcccgatc cgtcgacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5761 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5821 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5881 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5941 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6001 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6061 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6121 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6181 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6241 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6301 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6361 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6421 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6481 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6541 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     6601 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6661 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6721 caagtttact c
//
