LOCUS       sequence-430916-        7106 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 7106)
  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 7106)
  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: NPFF2-DuET
FEATURES             Location/Qualifiers
     source          1..7106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     325..344
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(563..667)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(742..761)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        933..1312
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1313..1516
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1467..1487
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1561..1580
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1561..1579
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3395..3412)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3401..3625
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3671..4099
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3758..3777)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3968..3989
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4108..4128)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4113..4442
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4293..4428
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4355..4374
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4509..5303
                     /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(4563..4582)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5173..5192
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5477..5598
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5514..5533)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5568..5587
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5647..5663)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5647..5663)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5660..5682)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5671..5687
                     /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(5695..5701,5702..5719,5720..5725))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5740..5761
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5878..5895)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6049..6637)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6129..6148)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6808..7106,1..493,494..562))
                     /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 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
       61 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
      121 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
      181 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
      241 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
      301 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
      361 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
      421 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
      481 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
      541 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
      601 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
      661 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
      721 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
      781 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
      841 aaggcaaggc ttgaccgaca tttgcatgaa gaatctgctt agggttaggc gttttgcgct
      901 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
      961 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     1021 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     1081 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     1141 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     1201 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     1261 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     1321 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     1381 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     1441 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     1501 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
     1561 taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa gcttagcggc
     1621 cgcgccacca tgaagacgat catcgccctg agctacatct tctgcctggt attcgccgac
     1681 tacaaggacg atgatgacgc cagcatcgat atgaatagtt tctttggcac acccgctgcg
     1741 tcatggtgcc tgcttgagtc tgacgtttcc agtgctcctg ataaggaggc cgggagagag
     1801 cgcagggccc ttagtgtgca acagagaggt ggaccagcat ggagtggttc acttgagtgg
     1861 tccaggcaga gcgctggcga taggcgacgc ctggggctgt cccgacaaac ggcgaagagc
     1921 tcctggtcca gaagcaggga cagaacatgt tgttgccggc gggcctggtg gattctcgtt
     1981 cctgctgctg atagggccag acgagaaaga tttattatga atgaaaagtg ggataccaat
     2041 tctagtgaaa actggcaccc aatctggaat gtgaatgaca caaagcatca tctttattcc
     2101 gatattaata taacgtatgt aaactattat ctgcatcagc cacaagtggc cgctatcttt
     2161 atcatcagct actttctgat tttctttctt tgcatgatgg gaaacaccgt tgtttgcttc
     2221 atcgtgatgc gaaataagca tatgcatacc gtgaccaact tgtttatcct gaacttggca
     2281 atcagtgatt tgctcgtagg aatattctgc atgcccatta ctctgctcga taatattatc
     2341 gcagggtggc ccttcggaaa cactatgtgc aagatctctg ggttggttca aggtatcagc
     2401 gtagctgcgt ccgtgttcac actcgttgcc atcgcagtgg atcgcttcca gtgcgtcgtg
     2461 tacccattta aacccaaact gactataaag actgcatttg tgatcattat gattatttgg
     2521 gtgcttgcca ttaccattat gtcacccagt gcagtcatgc tgcacgtgca ggaagagaag
     2581 tattaccgcg tccgactgaa ttcccagaac aaaacgagtc cagtgtactg gtgtcgggag
     2641 gactggccca accaggaaat gagaaaaatc tacaccacag tgctgtttgc caacatctac
     2701 ctcgcccctc tctccttgat tgttatcatg tacggtcgga ttggcatttc cctcttcagg
     2761 gcagctgttc cccacacagg aaggaagaac caggaacagt ggcacgtcgt gtcacggaag
     2821 aagcaaaaaa ttattaaaat gcttcttatc gtggctctcc tgttcatcct gtcatggctg
     2881 cccctttgga ccctgatgat gttgtccgac tacgccgacc tgtcccctaa cgagctccaa
     2941 attataaata tttatattta cccgtttgca cactggctgg cttttggcaa ttcctccgtt
     3001 aatcctatca tatacggatt tttcaacgaa aatttccgga gaggctttca agaagcgttt
     3061 cagctgcaac tgtgccaaaa gcgagcaaaa cccatggaag cctacgcgct caaggcaaag
     3121 agccatgtcc tcattaacac ttctaaccag ctcgttcagg agagcacttt tcagaatcct
     3181 cacggggaga cccttctgta taggaagtcc gctgaaaaac cgcaacaaga gttggtaatg
     3241 gaagagctga aagaaacaac caattcaagc gaaatcatcg ataccggtgg acgcaccggt
     3301 gaattcgatg aggccagcac caccgtgtct aagaccgaga cttctcaggt ggcccctgcc
     3361 taatctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     3421 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3481 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3541 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     3601 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     3661 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3721 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3781 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3841 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3901 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3961 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     4021 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     4081 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     4141 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     4201 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4261 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4321 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4381 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     4441 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     4501 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4561 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4621 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4681 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4741 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4801 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4861 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4921 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4981 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     5041 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     5101 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     5161 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     5221 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     5281 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     5341 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     5401 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     5461 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5521 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5581 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     5641 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5701 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5761 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5821 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5881 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5941 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     6001 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6061 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6121 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     6181 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     6241 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     6301 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6361 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6421 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6481 cactagaaga acggtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6541 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6601 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6661 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6721 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6781 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6841 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6901 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6961 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7021 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7081 tagttcgcca gttaatagtt tgcgca
//
