LOCUS       sequence-430744-        6935 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 6935)
  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 6935)
  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: ADRA2A-DuET
FEATURES             Location/Qualifiers
     source          1..6935
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     398..417
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(636..740)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(815..834)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1006..1385
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1386..1589
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1540..1560
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1634..1653
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1634..1652
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3297..3314)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3303..3527
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3573..4001
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3660..3679)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3870..3891
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4010..4030)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4015..4344
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4195..4330
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4257..4276
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4411..5205
                     /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(4465..4484)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5075..5094
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5379..5500
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5416..5435)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5470..5489
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5549..5565)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5549..5565)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5562..5584)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5573..5589
                     /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(5597..5603,5604..5621,5622..5627))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5642..5663
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5780..5797)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5951..6539)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6031..6050)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6710..6935,1..566,567..635))
                     /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 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
       61 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
      121 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
      181 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
      241 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
      301 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
      361 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
      421 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
      481 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
      541 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
      601 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
      661 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
      721 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
      781 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
      841 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
      901 tttaagctac aacaaggcaa ggcttgaccg acatttgcat gaagaatctg cttagggtta
      961 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     1021 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     1081 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     1141 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     1201 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     1261 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     1321 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     1381 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     1441 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     1501 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     1561 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
     1621 ggcttatcga aattaatacg actcactata gggagaccca agctggctag cgtttaaact
     1681 taagcttagc gccaccatga agacgatcat cgccctgagc tacatcttct gcctggtatt
     1741 cgccgactac aaggacgatg atgacgccag catcgatatg ttccggcagg agcagccact
     1801 cgccgagggt tcctttgccc ccatgggaag tcttcagcct gacgccggca acgctagctg
     1861 gaatggcacc gaagcgcccg gtggaggggc tcgggctacc ccatattctc tccaggtcac
     1921 actgaccctg gtttgtctgg ccggtctcct gatgctgctg acggtctttg gcaatgtttt
     1981 ggtgataatt gcggtgttca cctccagagc actcaaggct ccccagaacc tgttcttggt
     2041 cagcctggcg tctgccgata ttctggtcgc caccctcgta attccgtttt ctcttgccaa
     2101 tgaggttatg ggttactggt attttggtaa ggcatggtgc gagatatacc ttgctctcga
     2161 cgttctcttc tgcaccagca gtattgtgca tctgtgcgca attagtctgg acagatattg
     2221 gtccatcacg caggcaatag aatataactt gaaaaggact cctcggcgca ttaaggctat
     2281 catcatcact gtctgggtca ttagcgctgt gataagcttc cctccactta tttccattga
     2341 aaaaaaagga ggaggcggag ggccgcagcc tgcggagccc cgctgtgaga tcaacgatca
     2401 gaaatggtat gtcatctcct cctgcattgg gtccttcttc gccccatgtc tgatcatgat
     2461 cctcgtatat gttaggattt atcagatagc taagcgaagg acccgggtcc ccccatccag
     2521 gagaggtcca gacgcagtgg ccgctccccc cggaggcaca gaaaggagac ctaacggtct
     2581 gggaccagag cgctccgcag gacctggagg tgcagaagcc gagcccttgc cgacacaact
     2641 gaacggagct cccggcgaac cagccccagc aggtcctcgc gatactgacg cactggacct
     2701 cgaagagtcc tctagcagcg atcatgcaga gcgccctcca gggcctagga gacctgaaag
     2761 aggacccaga ggtaaaggca aggcccgggc aagtcaagtg aaaccgggag actctctgcc
     2821 tcggaggggt ccaggtgcta ctggtatagg gactcctgcc gccggtcccg gagaagagcg
     2881 ggtcggcgcc gctaaggcaa gtcgctggcg gggcagacaa aaccgggaaa aaaggtttac
     2941 attcgtgctc gcggtagtga ttggcgtctt cgtcgtgtgc tggtttccat tcttttttac
     3001 ctataccctg acagccgtcg gctgttctgt tccacggaca ctgttcaagt tctttttctg
     3061 gtttggctac tgtaactcaa gcctgaaccc ggtaatctat acaatattta accacgattt
     3121 tcggagggct tttaaaaaga tcctgtgcag aggcgatagg aaacgaatcg ttatcgatac
     3181 cggtggacgc accggtgaat tcgatgaggc cagcaccacc gtgtctaaga ccgagacttc
     3241 tcaggtggcc cctgcctaac tcgagtctag agggcccgtt taaacccgct gatcagcctc
     3301 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3361 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3421 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3481 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     3541 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     3601 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3661 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3721 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3781 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3841 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3901 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3961 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     4021 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4081 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4141 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     4201 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4261 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     4321 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     4381 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     4441 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     4501 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     4561 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     4621 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4681 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4741 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4801 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4861 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4921 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4981 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     5041 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     5101 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     5161 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     5221 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     5281 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5341 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5401 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5461 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5521 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5581 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5641 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5701 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5761 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5821 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5881 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5941 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6001 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6061 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6121 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6181 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6241 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6301 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6361 agtggtggcc taactacggc tacactagaa gaacggtatt tggtatctgc gctctgctga
     6421 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6481 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6541 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6601 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6661 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6721 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6781 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6841 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6901 gaagggccga gcgcagaagt ggtcctgcaa cttta
//
