LOCUS       sequence-430875-        6626 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 6626)
  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 6626)
  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: MTNR1B-DuET
FEATURES             Location/Qualifiers
     source          1..6626
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     442..461
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(680..784)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(859..878)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1050..1429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1430..1633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1584..1604
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1678..1697
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1678..1696
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3032..3049)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3038..3262
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3308..3736
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3395..3414)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3605..3626
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3745..3765)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3750..4079
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3930..4065
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3992..4011
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4146..4940
                     /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(4200..4219)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4810..4829
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5114..5235
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5151..5170)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5205..5224
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5284..5300)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5284..5300)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5297..5319)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5308..5324
                     /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(5332..5338,5339..5356,5357..5362))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5377..5398
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5515..5532)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5686..6274)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5766..5785)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6445..6626,1..610,611..679))
                     /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 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
       61 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
      121 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
      181 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
      241 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
      301 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
      361 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
      421 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
      481 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
      541 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
      601 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
      661 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
      721 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
      781 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
      841 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
      901 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
      961 gcaaggcttg accgacattt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     1021 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     1081 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     1141 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     1201 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     1261 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     1321 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     1381 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     1441 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     1501 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     1561 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     1621 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
     1681 tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct tagcgccacc
     1741 atgaagacga tcatcgccct gagctacatc ttctgcctgg tattcgccga ctacaaggac
     1801 gatgatgacg ccagcatcga tatgagcgag aacggttcct tcgctaattg ctgcgaagca
     1861 ggtggatggg ccgttcgccc tgggtggtcc ggagcaggtt ctgcaagacc ctcaagaact
     1921 ccacgcccac cttgggtggc acctgctctt tccgccgtgc tcattgtcac aactgccgta
     1981 gatgttgttg ggaatctttt ggtcattctc tctgtactga ggaatcggaa gttgcggaat
     2041 gcaggcaacc tcttcctcgt gtcactggca cttgctgacc ttgtggtggc cttctatcca
     2101 taccccttga tactggttgc tatcttctac gatggctggg ctcttggtga ggagcactgt
     2161 aaggccagcg ctttcgtgat ggggttgtcc gtcattggaa gtgtttttaa cattacagca
     2221 atagctatca atcggtactg ctatatttgt cacagcatgg cataccatag aatctacaga
     2281 cgctggcata ctccactcca catttgcctt atatggcttc tcacagttgt ggccctgttg
     2341 cctaactttt tcgttggatc cttggagtac gacccccgaa tttactcatg cacctttata
     2401 caaaccgcca gcacccagta tactgctgca gtcgtggtca tccatttcct tctccctatt
     2461 gccgtcgttt cattttgcta tctccggatc tgggtgttgg tcctgcaggc taggcggaag
     2521 gcaaaacccg agtctcggct ttgcctgaag ccaagcgacc tgcgatcttt tttgaccatg
     2581 ttcgtcgtct tcgtgatttt cgccatctgt tgggctccac tgaattgcat cggcctggct
     2641 gttgcgatca atccacaaga aatggcacct caaattcccg aggggctctt cgtgaccagc
     2701 taccttctgg catatttcaa cagttgcctg aatgctatcg tgtacggatt gctgaaccag
     2761 aacttccgaa gggaatataa aaggatcctg ctggcactct ggaaccctag gcattgtatc
     2821 caagacgcat ccaaggggtc tcacgcggaa ggcctccaga gccctgcacc acccattatt
     2881 ggtgtgcaac atcaggccga cgctttgatc gataccggtg gacgcaccgg tgaattcgat
     2941 gaggccagca ccaccgtgtc taagaccgag acttctcagg tggcccctgc ctaactcgag
     3001 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     3061 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     3121 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     3181 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     3241 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     3301 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     3361 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     3421 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     3481 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     3541 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3601 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3661 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3721 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     3781 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3841 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3901 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3961 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     4021 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     4081 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     4141 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     4201 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     4261 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     4321 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     4381 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     4441 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     4501 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     4561 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     4621 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     4681 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     4741 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     4801 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     4861 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4921 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     4981 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     5041 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     5101 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     5161 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     5221 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     5281 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5341 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5401 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5461 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5521 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5581 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5641 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5701 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5761 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5821 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5881 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5941 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6001 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6061 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6121 tagaagaacg gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6181 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6241 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6301 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6361 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6421 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6481 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6541 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6601 ggctccagat ttatcagcaa taaacc
//
