LOCUS       sequence-430856-        6635 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 6635)
  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 6635)
  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: HTR1E-DuET
FEATURES             Location/Qualifiers
     source          1..6635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     573..592
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(811..915)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(990..1009)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1181..1560
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1561..1764
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1715..1735
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1809..1828
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1809..1827
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3172..3189)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3178..3402
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3448..3876
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3535..3554)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3745..3766
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3885..3905)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3890..4219
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4070..4205
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4132..4151
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4286..5080
                     /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(4340..4359)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4950..4969
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5254..5375
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5291..5310)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5345..5364
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5424..5440)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5424..5440)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5437..5459)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5448..5464
                     /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(5472..5478,5479..5496,5497..5502))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5517..5538
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5655..5672)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5826..6414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5906..5925)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6585..6635,1..741,742..810))
                     /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 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
       61 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
      121 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
      181 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
      241 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
      301 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
      361 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
      421 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
      481 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
      541 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
      601 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
      661 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
      721 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
      781 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
      841 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
      901 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
      961 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     1021 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     1081 gctacaacaa ggcaaggctt gaccgacatt tgcatgaaga atctgcttag ggttaggcgt
     1141 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
     1201 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     1261 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     1321 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     1381 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     1441 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     1501 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     1561 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     1621 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     1681 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     1741 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
     1801 atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt aaacttaagc
     1861 ttagcgccac catgaagacg atcatcgccc tgagctacat cttctgcctg gtattcgccg
     1921 actacaagga cgatgatgac gccagcatcg atatgaatat aactaactgc actacagagg
     1981 cctctatggc tatcaggccc aagactatca ctgagaagat gctcatctgc atgaccctcg
     2041 tcgtgatcac cacactgact acactcctca atctggcagt gatcatggca atcggcacta
     2101 cgaagaagct tcatcagccg gctaactatc tgatttgcag cctggcggtg accgatctgc
     2161 tcgtggcagt cctggtgatg ccattgtcta taatttatat cgtgatggat cgctggaaac
     2221 ttggttactt tctgtgcgag gtgtggctgt ctgtcgatat gacttgctgc acctgcagta
     2281 tcctgcatct ctgcgtgatc gcactcgaca gatactgggc cataacgaat gcaattgaat
     2341 atgcgaggaa aagaaccgct aagcgcgcag ccttgatgat cctgaccgtg tggacaatta
     2401 gtattttcat atccatgccc cccctgttct ggagatccca ccggcgcctg tccccacccc
     2461 cttctcagtg caccatacag catgaccatg tgatttacac tatctactcc actctgggcg
     2521 ccttctatat tccactgact ctgatcttga ttctctacta tcggatctac cacgctgcta
     2581 agtctctgta ccagaagcgg ggctcatcac ggcatctgag taacagatct actgacagcc
     2641 agaattcttt cgcatcttgc aagctcacac agacgttttg tgtctcagac ttcagtacat
     2701 ccgaccctac caccgaattt gaaaaattcc acgcctcaat ccgaattccc ccttttgaca
     2761 atgatctgga ccaccctgga gaaagacaac aaattagttc tacccgggag cgaaaggcgg
     2821 ccaggatcct gggactgatc ctcggagctt ttattctctc atggctccct tttttcatta
     2881 aggaactcat cgtgggcctt agtatctata ccgtctcttc cgaggtagcg gacttcctta
     2941 cctggctggg atatgttaac agcctcatta accccctgct ctacactagc tttaatgagg
     3001 acttcaagct ggctttcaag aagcttatac gctgtagaga gcatacaatc gataccggtg
     3061 gacgcaccgg tgaattcgat gaggccagca ccaccgtgtc taagaccgag acttctcagg
     3121 tggcccctgc ctaactcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     3181 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3241 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3301 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3361 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3421 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3481 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3541 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3601 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3661 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3721 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3781 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3841 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3901 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3961 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4021 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4081 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4141 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     4201 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     4261 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4321 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4381 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4441 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     4501 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4561 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4621 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4681 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4741 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4801 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4861 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4921 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4981 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     5041 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     5101 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     5161 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     5221 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     5281 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     5341 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     5401 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5461 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5521 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5581 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5641 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5701 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5761 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5821 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5881 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5941 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6001 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6061 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6121 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6181 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6241 tggcctaact acggctacac tagaagaacg gtatttggta tctgcgctct gctgaagcca
     6301 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6361 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6421 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6481 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6541 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6601 agtgaggcac ctatctcagc gatctgtcta tttcg
//
