LOCUS       sequence-430878-        6665 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 6665)
  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 6665)
  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: NPY4R-DuET
FEATURES             Location/Qualifiers
     source          1..6665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     213..232
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(451..555)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(630..649)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        821..1200
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1201..1404
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1355..1375
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1449..1468
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1449..1467
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2842..2859)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2848..3072
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3118..3546
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3205..3224)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3415..3436
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3555..3575)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3560..3889
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3740..3875
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3802..3821
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3956..4750
                     /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(4010..4029)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4620..4639
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4924..5045
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4961..4980)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5015..5034
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5094..5110)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5094..5110)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5107..5129)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5118..5134
                     /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(5142..5148,5149..5166,5167..5172))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5187..5208
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5325..5342)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5496..6084)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5576..5595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6255..6665,1..381,382..450))
                     /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 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
       61 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
      121 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
      181 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
      241 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
      301 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
      361 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
      421 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
      481 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
      541 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
      601 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
      661 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
      721 gctacaacaa ggcaaggctt gaccgacatt tgcatgaaga atctgcttag ggttaggcgt
      781 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
      841 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      901 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      961 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     1021 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     1081 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     1141 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     1201 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     1261 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     1321 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     1381 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
     1441 atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt aaacttaagc
     1501 ttagcgccac catgaagacg atcatcgccc tgagctacat cttctgcctg gtattcgccg
     1561 actacaagga cgatgatgac gccagcatcg atatgaacac ttcacacctg ctggccctgt
     1621 tgcttccaaa gagcccacaa ggcgagaatc gcagcaagcc cttggggact ccttataact
     1681 tctcagagca ctgtcaggac tccgtggatg tgatggtatt catcgtcacg tcctactcca
     1741 tcgaaacagt cgtgggagtg ctcgggaacc tctgtctcat gtgtgtgact gtccggcaaa
     1801 aagaaaaggc taacgtcacc aacctgctga ttgctaacct ggcattcagc gatttcctta
     1861 tgtgcttgct gtgccagcca ctgacagcgg tttacaccat catggactat tggatattcg
     1921 gagagacact gtgtaagatg agcgctttta tacagtgtat gtcagttact gtgagcattc
     1981 tgtctttggt ccttgtcgcc ctcgaaaggc atcagttgat tattaatccc acgggctgga
     2041 agccctctat ctctcaagca tatctgggta tcgtgctgat ctgggtcatt gcatgcgtgc
     2101 ttagtctccc cttcctggct aacagtattt tggagaacgt ctttcacaag aaccatagca
     2161 aagctctgga gtttttggcc gacaaggtcg tatgcactga gtcctggcca ttggcacatc
     2221 accggacaat ttatacaacc ttcctgctgt tgttccagta ctgtctgccc ctcggtttta
     2281 tcctcgtctg ctatgccagg atctacaggc gcctgcagcg ccaggggcgc gtgttccaca
     2341 aagggaccta ttccctcagg gctggacaca tgaaacaggt gaatgttgtt ctcgtcgtga
     2401 tggtggttgc ttttgcggtg ttgtggctgc cactccacgt gttcaacagc ctcgaagatt
     2461 ggcatcacga ggccatccca atctgtcacg gtaatctgat tttcctcgtc tgtcatctct
     2521 tggccatggc ttctacctgc gtgaacccct ttatatacgg ctttttgaat acaaatttca
     2581 agaaggagat caaggcactc gtgcttacat gtcagcagag cgccccgctg gaggagtccg
     2641 aacacttgcc cctgtccact gtgcacacgg aagtcagcaa gggatccttg aggctgagcg
     2701 ggcgcagtaa cccgataatc gataccggtg gacgcaccgg tgaattcgat gaggccagca
     2761 ccaccgtgtc taagaccgag acttctcagg tggcccctgc ctaactcgag tctagagggc
     2821 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     2881 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     2941 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     3001 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     3061 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     3121 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3181 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3241 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3301 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     3361 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3421 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3481 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3541 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     3601 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     3661 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3721 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3781 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     3841 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     3901 agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat
     3961 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4021 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4081 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     4141 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4201 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4261 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4321 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4381 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4441 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     4501 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4561 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4621 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4681 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4741 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4801 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4861 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4921 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4981 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     5041 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     5101 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5161 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5221 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5281 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5341 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5401 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5461 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5521 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5581 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5641 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5701 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5761 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5821 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5881 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaacg
     5941 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6001 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6061 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6121 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6181 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6241 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6301 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6361 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6421 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6481 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6541 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6601 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6661 ttgtg
//
