LOCUS       sequence-431819-        6854 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 6854)
  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 6854)
  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: HTR6-DuET
FEATURES             Location/Qualifiers
     source          1..6854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     438..457
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(676..780)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(855..874)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1046..1425
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1426..1629
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1580..1600
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1674..1693
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1674..1692
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3256..3273)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3262..3486
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3532..3960
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3619..3638)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3829..3850
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3969..3989)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3974..4303
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4154..4289
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4216..4235
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4370..5164
                     /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(4424..4443)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5034..5053
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5338..5459
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5375..5394)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5429..5448
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5508..5524)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5508..5524)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5521..5543)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5532..5548
                     /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(5556..5562,5563..5580,5581..5586))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5601..5622
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5739..5756)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5910..6498)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5990..6009)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6669..6854,1..606,607..675))
                     /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 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
       61 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
      121 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
      181 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
      241 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
      301 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
      361 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
      421 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
      481 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
      541 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
      601 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
      661 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
      721 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
      781 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
      841 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
      901 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
      961 ggcttgaccg acatttgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     1021 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
     1081 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     1141 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     1201 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     1261 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     1321 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     1381 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     1441 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     1501 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     1561 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     1621 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg
     1681 actcactata gggagaccca agctggctag cgtttaaact taagcttagc ggccgcgcca
     1741 ccatgaagac gatcatcgcc ctgagctaca tcttctgcct ggtattcgcc gactacaagg
     1801 acgatgatga cgccagcatc gatatggtgc ccgagccggg cccaactgct aacagcaccc
     1861 cagcttgggg ggctggtcct ccaagcgccc caggtggcag tggttgggtt gctgcagcat
     1921 tgtgcgtggt tatcgccctg acagctgccg caaacagcct gctcattgct cttatttgta
     1981 cccaacccgc actccggaat acgtctaatt ttttcctggt ctcactgttc acgagcgacc
     2041 ttatggtcgg gctcgtagtc atgcctcctg ctatgttgaa cgccctgtat gggagatggg
     2101 tcctggcaag gggtctttgc cttctgtgga ccgcatttga cgtcatgtgc tgtagtgcca
     2161 gcatcttgaa cctctgtttg attagtctgg atagatatct gctgattctg agcccactta
     2221 gatacaagct gcggatgact ccgctccggg ccctcgcttt ggtgctgggc gcctggtctc
     2281 tggcagcact ggcttcattc ttgcctttgc tgctgggctg gcacgaattg gggcacgcaa
     2341 ggccgccagt tccaggccag tgtaggctgc ttgcctccct gcccttcgtg ttggtggctt
     2401 ccggcctcac attctttctg cctagtggtg ccatctgctt cacttactgc cggatacttc
     2461 tggcagctag aaaacaagcc gttcaggttg cctccctcac caccggcatg gcaagccagg
     2521 cctctgagac cctgcaggtg ccgaggactc ctaggcctgg cgtagagtct gccgactcac
     2581 ggcggctggc cactaaacac tcaagaaagg cgctgaaagc ttcattgacc ttgggcatct
     2641 tgctgggcat gttcttcgta acatggctcc cattttttgt ggccaatatt gtccaggcgg
     2701 tgtgcgactg catctcacca ggcctgttcg atgtgctcac ttggctgggg tattgcaatt
     2761 ccactatgaa ccccatcata tatcccctgt tcatgagaga tttcaagcga gcactcggaa
     2821 ggtttctgcc atgcccacgg tgcccacgcg aaaggcaagc aagtctggcc tccccctcac
     2881 tccggacctc ccattcagga cccagaccag gactttcact ccagcaagtt ctgcccctgc
     2941 cacttcctcc agattcagac tctgactccg atgcgggttc aggtggctct agcggactcc
     3001 ggcttacggc ccagcttttg cttcccgggg aagcaactca ggacccacca ttgcctactc
     3061 gggctgccgc cgccgtcaat ttcttcaaca ttgatcctgc agagccagaa ctcagaccgc
     3121 atcctctggg tataccaacc aatatcgata ccggtgaatt cgatgaggcc agcaccaccg
     3181 tgtctaagac cgagacttct caggtggccc ctgcctaact cgagtctaga gggcccgttt
     3241 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3301 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3361 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3421 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     3481 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3541 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3601 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3661 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3721 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     3781 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3841 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3901 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3961 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     4021 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     4081 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     4141 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4201 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     4261 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     4321 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4381 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4441 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4501 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     4561 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4621 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4681 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4741 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4801 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4861 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     4921 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4981 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     5041 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     5101 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     5161 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     5221 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     5281 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     5341 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     5401 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     5461 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     5521 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5581 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5641 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5701 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5761 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5821 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5881 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5941 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6001 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6061 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6121 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6181 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6241 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6301 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacggtattt
     6361 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6421 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6481 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6541 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6601 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6661 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6721 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6781 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6841 gcaataaacc agcc
//
