LOCUS       sequence-430855-        6671 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 6671)
  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 6671)
  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: HTR1D-DuET
FEATURES             Location/Qualifiers
     source          1..6671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     445..464
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(585..914)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      599..734
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(653..672)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     899..919
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(928..1356)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1038..1059)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1250..1269
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1402..1626
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1615..1632
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(3012..3031)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3013..3031)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3076..3279)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3105..3125)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3280..3659
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3831..3850
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3925..4029
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4030..4098,4099..4890)
                     /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"
     primer_bind     complement(4248..4267)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5061..5649
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5550..5569
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5803..5820
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5937..5958
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5973..5978,5979..5996,5997..6003)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6011..6027
                     /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)."
     primer_bind     6016..6038
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6035..6051
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6035..6051
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    6100..6221
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6111..6130)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6165..6184
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(6395..6671,1..518))
                     /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(6506..6525)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg
       61 acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg
      121 aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat
      181 actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat
      241 agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc
      301 gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac
      361 aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca
      421 tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg
      481 gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc
      541 tcttgatcag atccgaaaat ggatatacaa gctcccggga gctttttgca aaagcctagg
      601 cctccaaaaa agcctcctca ctacttctgg aatagctcag aggcagaggc ggcctcggcc
      661 tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga
      721 gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc
      781 atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac
      841 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
      901 cctaactgac acacattcca cagaattaat tcgcgttaaa tttttgttaa atcagctcat
      961 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     1021 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     1081 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     1141 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     1201 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     1261 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     1321 cacccgccgc gcttaatgcg ccgctacagg gcgcgtgggg atacccccta gagccccagc
     1381 tggttctttc cgcctcagaa gccatagagc ccaccgcatc cccagcatgc ctgctattgt
     1441 cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc
     1501 tactcagaca atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac
     1561 cttccagggt caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag
     1621 gcacagtcga ggctgatcag cgggtttaaa cgggccctct agactcgagt taggcagggg
     1681 ccacctgaga agtctcggtc ttagacacgg tggtgctggc ctcatcgaat tcaccggtgc
     1741 gtccaccggt atcgatagag gcctttctaa aagggacgat cttctgaaag gcctgtcgga
     1801 actcctcatt gaacacagtg tagatgatag ggttgataag actattcaga tagcccagcc
     1861 aggtgaagaa gtcgaacaag gctggatgaa tccagcagct atctctacaa ataggaagta
     1921 cgagggacac aacaaaaaaa ggcagccagc aaatgataaa ggctccgagg attattccca
     1981 gaatcttcgt ggccttccgt tcccgcgcag cagatattct cttgcgttcc agagctgagt
     2041 cggccagctt aattttcaca tggttaaaaa aaagtggtga cccagcggag tgagagtgcc
     2101 cctcgtggag tgaagagttc agagagcaca ggctgcttcc agcgctccca gtaatcaagt
     2161 gagctgtcgt aaatctcttt ccatacaggg acggtggatt caaaatgcgg ttcctggcgg
     2221 ctcggtagat ccggccatac aagatgatga gcagaacgga tggaatatag aaggcaccac
     2281 atgtactata gatggtataa ctgatctggc tggtgttcac caggcagtcg ctcatctcct
     2341 cctgtgcctt agcctgcctc cagaagaggg gggggatgga gatacaaatg gaaatggccc
     2401 atacgatggc aatcatagtt gcggcatgtc ccgccgtgcg cctcttgctg tactccaggg
     2461 cgtcggtaat agcccagtat cggtcaagtg caatgacaca caggtggaga atagaggcag
     2521 tacaacaggt gatgtcagag ctcagccaaa tgtcacacag gatctgcccg aaattccagg
     2581 tatgcgtaat tgtatacgca atggagatag gcatcacaag gatactcacc aacaaatctg
     2641 tggtggcaag ggagccaatc aaataattag ctggggtgtg cagcttccga gtgagcagga
     2701 tggttgtcaa cacaaatgca ttggagagaa ctgtggcgag cgtaatcaca gacaggacaa
     2761 cagccaggga aattttgagg gcctgcagtg tgcggggatc ccaggcttcg ctcgtctcag
     2821 ttgcattcaa ggaccgattg ctggcctcct gtggcagccc ctcagcactc tggttaagag
     2881 gactcatatc gatgctggcg tcatcatcgt ccttgtagtc ggcgaatacc aggcagaaga
     2941 tgtagctcag ggcgatgatc gtcttcatgg tggcgctaag cttaagttta aacgctagcc
     3001 agcttgggtc tccctatagt gagtcgtatt aatttcgata agccagtaag cagtgggttc
     3061 tctagttagc cagagagctc tgcttatata gacctcccac cgtacacgcc taccgcccat
     3121 ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca
     3181 aaacaaactc ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg
     3241 ctatccacgc ccattgatgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa
     3301 tacgtagatg tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca
     3361 ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg
     3421 atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt
     3481 ccctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt
     3541 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg
     3601 gctatgaact aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca
     3661 acgcgtatat ctggcccgta catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt
     3721 cttcatgcaa atgtcggtca agccttgcct tgttgtagct taaattttgc tcgcgcacta
     3781 ctcagcgacc tccaacacac aagcagggag cagatactgg cttaactatg cggcatcaga
     3841 gcagattgta ctgagagtgc accatagggg atcgggagat ctcccgatcc gtcgacgtca
     3901 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3961 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     4021 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     4081 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     4141 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     4201 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     4261 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     4321 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     4381 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     4441 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     4501 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     4561 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     4621 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     4681 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     4741 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     4801 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     4861 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     4921 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4981 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     5041 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5101 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5161 tttccgaagg taactggctt cagcagagcg cagataccaa ataccgttct tctagtgtag
     5221 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5281 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5341 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5401 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     5461 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     5521 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     5581 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     5641 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     5701 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     5761 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     5821 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     5881 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     5941 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     6001 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     6061 gccaagctct agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg
     6121 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     6181 ttgctttatt tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc
     6241 agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag
     6301 cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc
     6361 gcttggtcgg tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat
     6421 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag
     6481 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc
     6541 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca
     6601 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc
     6661 gcgccttgag c
//
