LOCUS       sequence-430874-        6590 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 6590)
  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 6590)
  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: MTNR1A-DuET
FEATURES             Location/Qualifiers
     source          1..6590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     460..479
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(698..802)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(877..896)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1068..1447
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1448..1651
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1602..1622
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1696..1715
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1696..1714
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3014..3031)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3020..3244
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3290..3718
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3377..3396)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3587..3608
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3727..3747)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3732..4061
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3912..4047
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3974..3993
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4128..4922
                     /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(4182..4201)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4792..4811
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5096..5217
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5133..5152)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5187..5206
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5266..5282)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5266..5282)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5279..5301)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5290..5306
                     /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(5314..5320,5321..5338,5339..5344))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5359..5380
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5497..5514)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5668..6256)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5748..5767)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6427..6590,1..628,629..697))
                     /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"
ORIGIN
        1 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
       61 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
      121 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
      181 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
      241 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
      301 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
      361 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
      421 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
      481 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
      541 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
      601 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
      661 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
      721 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
      781 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
      841 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
      901 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
      961 aatttaagct acaacaaggc aaggcttgac cgacatttgc atgaagaatc tgcttagggt
     1021 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
     1081 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     1141 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     1201 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     1261 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     1321 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     1381 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     1441 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     1501 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     1561 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     1621 tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac ccactgctta
     1681 ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct agcgtttaaa
     1741 cttaagctta gcgccaccat gaagacgatc atcgccctga gctacatctt ctgcctggta
     1801 ttcgccgact acaaggacga tgatgacgcc agcatcgata tgcagggcaa cggttcagca
     1861 ctcccgaatg caagccagcc agtgctgcgg ggagacggcg cccgacctag ttggttggcc
     1921 agcgccctgg cctgcgtcct tatcttcacc atcgtggtcg acatcctggg caatctgctg
     1981 gtcatcctgt cagtttatag gaacaaaaag ctgcggaatg ccggcaacat atttgtggtc
     2041 tcactcgctg tagcggatct tgttgtcgcc atctacccat atccactcgt gctcatgagt
     2101 atttttaata acggttggaa cctgggttac ttgcattgtc aagtgagcgg atttttgatg
     2161 ggcctgtctg tcatcggaag catattcaat atcacaggca ttgccatcaa caggtattgc
     2221 tacatatgtc acagtctgaa atacgacaag ttgtatagct caaagaatag cctgtgttac
     2281 gtcctgctga tatggctgct gacccttgct gccgtcctgc cgaatctgag ggcaggcacc
     2341 ctgcaatacg atcccagaat ttattcatgt actttcgctc agagcgtgtc tagcgcctac
     2401 accattgccg ttgtggtttt ccattttctg gtgccaatga tcattgtcat cttttgttat
     2461 cttcgcatat ggattcttgt gcttcaggtg cggcagagag tgaaacccga ccgaaagcca
     2521 aagttgaagc cccaggattt ccggaacttc gttaccatgt ttgtcgtctt cgtactgttc
     2581 gcaatctgct gggcccctct gaatttcatt ggcctggcgg tggcctcaga ccccgccagt
     2641 atggttccta gaatacctga gtggctgttc gtagcgagct actacatggc ttactttaac
     2701 tcttgtctta acgctatcat ctacggactg cttaaccaga acttcaggaa ggagtacagg
     2761 agaatcattg tttctctttg taccgcacgc gtattctttg tagacagctc caatgacgtg
     2821 gcagatcgag tcaaatggaa gcctagccct ctgatgacta ataataacgt cgtgaaagtc
     2881 gacagtgtga tcgataccgg tggacgcacc ggtgaattcg atgaggccag caccaccgtg
     2941 tctaagaccg agacttctca ggtggcccct gcctaactcg agtctagagg gcccgtttaa
     3001 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     3061 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     3121 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     3181 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     3241 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     3301 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3361 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3421 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3481 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3541 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3601 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3661 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     3721 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3781 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3841 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3901 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3961 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     4021 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     4081 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     4141 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     4201 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     4261 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     4321 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     4381 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4441 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4501 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4561 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4621 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     4681 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4741 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4801 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4861 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4921 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4981 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     5041 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     5101 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5161 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5221 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     5281 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5341 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5401 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5461 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5521 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5581 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5641 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5701 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5761 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5821 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5881 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5941 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6001 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6061 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cggtatttgg
     6121 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6181 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6241 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6301 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6361 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6421 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6481 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6541 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
//
