LOCUS       sequence-430847-        6701 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 6701)
  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 6701)
  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: HCA3-DuET
FEATURES             Location/Qualifiers
     source          1..6701
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     412..431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(650..754)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(829..848)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1020..1399
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1400..1603
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1554..1574
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1648..1667
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1648..1666
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3077..3094)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3083..3307
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3353..3781
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3440..3459)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3650..3671
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3790..3810)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3795..4124
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3975..4110
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4037..4056
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4191..4985
                     /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(4245..4264)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4855..4874
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5159..5280
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5196..5215)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5250..5269
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5329..5345)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5329..5345)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5342..5364)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5353..5369
                     /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(5377..5383,5384..5401,5402..5407))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5422..5443
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5560..5577)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5731..6319)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5811..5830)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6490..6701,1..580,581..649))
                     /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 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
       61 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
      121 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
      181 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
      241 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
      301 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
      361 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
      421 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
      481 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
      541 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
      601 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
      661 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
      721 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
      781 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
      841 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
      901 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacattt gcatgaagaa
      961 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
     1021 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     1081 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     1141 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     1201 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     1261 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     1321 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     1381 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     1441 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     1501 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     1561 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
     1621 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg
     1681 ctagcgttta aacttaagct tagcgccacc atgaagacga tcatcgccct gagctacatc
     1741 ttctgcctgg tattcgccga ctacaaggac gatgatgacg ccagcatcga tatgaatcgc
     1801 caccacctgc aggatcactt tctcgaaatc gacaagaaga actgttgcgt cttccgggac
     1861 gattttatag ccaaagtgct cccacccgtg ctgggcctgg agttcatctt tggcctcttg
     1921 ggcaacgggt tggccctttg gatcttctgc ttccatctca agagctggaa atcttcccgg
     1981 attttcctct tcaatttggc cgtagcagat ttcctgctca tcatctgtct tcctttcgtc
     2041 atggactact atgtgcgcag aagcgattgg aagtttggtg acattccctg ccgcttggtt
     2101 ttgtttatgt ttgctatgaa ccgacagggt agcatcatct ttctgaccgt cgttgccgtc
     2161 gaccgatatt ttcgggtggt gcatcctcac catgccttga acaaaatatc caactggaca
     2221 gccgctataa tttcttgctt gctgtggggc attactgtgg ggctgactgt gcaccttctg
     2281 aagaagaaat tgctgatcca gaatgggacc gcgaacgtat gcatttcttt ctctatatgt
     2341 catacatttc gatggcatga ggccatgttc ctgctggagt tttttcttcc actcgggatt
     2401 atccttttct gtagtgctag gatcatctgg agtttgcggc aacgccaaat ggatcggcat
     2461 gctaaaatta agcgagccat aacgttcatt atggtggttg ccatagtgtt cgtgatctgt
     2521 tttctgccta gcgtcgtcgt gaggatccac atcttttggc tgctgcacac ctctgggacc
     2581 cagaactgcg aagtgtaccg atcagtagat ctcgcgtttt ttatcacatt gtcctttact
     2641 tacatgaact ccatgctcga ccctgtggtt tactacttca gttctccctc ttttccaaac
     2701 ttcttttcca ccctgattaa tcggtgcctt caaaggaaga ttacaggaga gcctgataat
     2761 aacaggagca cttcagtcga gctgaccgga gatcccaaca agactcgggg agcccctgag
     2821 gcacttatcg ccaattctgg agaaccctgg agccctagct acctggggcc aacatctaat
     2881 aaccattcta agaaggggca ctgccaccag gagccagcca gcttggagaa acagttgggc
     2941 tgttgtatcg aaatcgatac cggtggacgc accggtgaat tcgatgaggc cagcaccacc
     3001 gtgtctaaga ccgagacttc tcaggtggcc cctgcctaac tcgagtctag agggcccgtt
     3061 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     3121 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3181 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3241 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3301 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     3361 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3421 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3481 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3541 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     3601 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3661 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     3721 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3781 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     3841 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     3901 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3961 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     4021 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     4081 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     4141 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     4201 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     4261 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     4321 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     4381 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     4441 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     4501 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     4561 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4621 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4681 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     4741 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4801 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4861 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     4921 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4981 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     5041 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     5101 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     5161 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     5221 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5281 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5341 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5401 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5461 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5521 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5581 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5641 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5701 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5761 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5821 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5881 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5941 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6001 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6061 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6121 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacggtatt
     6181 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6241 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6301 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6361 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6421 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6481 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6541 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6601 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6661 agcaataaac cagccagccg gaagggccga gcgcagaagt g
//
