LOCUS       sequence-430745-        6890 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 6890)
  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 6890)
  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: ADRA2B-DuET
FEATURES             Location/Qualifiers
     source          1..6890
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(848..867)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(849..867)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(912..1115)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(941..961)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1116..1495
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1667..1686
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1761..1865
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1866..1934,1935..2726)
                     /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(2084..2103)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2897..3485
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3386..3405
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3639..3656
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3773..3794
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3809..3814,3815..3832,3833..3839)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3847..3863
                     /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     3852..3874
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3871..3887
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3871..3887
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3936..4057
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3947..3966)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4001..4020
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4231..5025)
                     /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(4342..4361)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4952..4971
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5092..5421)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5106..5241
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5406..5426
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5435..5863)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5545..5566)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5757..5776
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5909..6133
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6122..6139
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 cgcgacggag gccagtgcgg gcagcttggc actggcaagt gcgccgccgt gatcaggcct
       61 aggctgcttg gattccccct ggcctggtcc tcccttagcc cgaggcccgc gtctattaga
      121 ccgcttagcg atcaggtaaa tcctgaggta taccaaaatc atgatcaggc atggggcaaa
      181 gaagcttccg atgctgctcg ccaaaatgta ccaagcctct tgattcagct tgcattgtgg
      241 ccgccctctg ggttgagggc cctgatctcc tttgtatata agggggggca ggcttattac
      301 tgccgctatg agccagactg taaggattat gcacttgatc ctcctaggtg tccgcttgct
      361 attatactcc agagcccttg acacggccca atatctgtcc aggctgattg cacaaagatg
      421 gacaattgaa gaggtgcaaa acagtacgtc cagggcgaga tacacctcgc accaggttcg
      481 gcgaaaatac caatatccca acaactcatt agcgagtgaa aagggaatga tgagggttgc
      541 gacgagaatg tcagctgccg cgagggagac cagaaaaagg ttttgtggtg ccctgagtga
      601 cctgctggtg agcaccgcca gaattacaag cgcgttcccg aagatggtaa agagaatgag
      661 aaaagtgata gcggcagcga tggccgcagt ggcctggaca gaatagggat cctgatgatc
      721 catatcgatg ctggcgtcat catcgtcctt gtagtcggcg aataccaggc agaagatgta
      781 gctcagggcg atgatcgtct tcatggtggc gctaagctta agtttaaacg ctagccagct
      841 tgggtctccc tatagtgagt cgtattaatt tcgataagcc agtaagcagt gggttctcta
      901 gttagccaga gagctctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc
      961 gtcaatgggg cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac
     1021 aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat
     1081 ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg
     1141 tagatgtact gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg
     1201 ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt
     1261 actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct
     1321 attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc
     1381 ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta
     1441 tgaactaatg accccgtaat tgattactat taataactag tcaataatca atgtcaacgc
     1501 gtatatctgg cccgtacatc gcgaagcagc gcaaaacgcc taaccctaag cagattcttc
     1561 atgcaaatgt cggtcaagcc ttgccttgtt gtagcttaaa ttttgctcgc gcactactca
     1621 gcgacctcca acacacaagc agggagcaga tactggctta actatgcggc atcagagcag
     1681 attgtactga gagtgcacca taggggatcg ggagatctcc cgatccgtcg acgtcaggtg
     1741 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1801 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     1861 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     1921 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     1981 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2041 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2101 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2161 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2221 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2281 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2341 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     2401 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     2461 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     2521 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     2581 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     2641 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     2701 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     2761 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     2821 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2881 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2941 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3001 cgaaggtaac tggcttcagc agagcgcaga taccaaatac cgttcttcta gtgtagccgt
     3061 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3121 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3181 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3241 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3301 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3361 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3421 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3481 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3541 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3601 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3661 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     3721 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     3781 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     3841 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     3901 agctctagct agaggtcgac ggtatacaga catgataaga tacattgatg agtttggaca
     3961 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     4021 tttatttgta accattataa gctgcaataa acaagttggg gtgggcgaag aactccagca
     4081 tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca
     4141 acctttcata gaaggcggcg gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt
     4201 ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa
     4261 ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca
     4321 ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc
     4381 cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat
     4441 attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc
     4501 cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc
     4561 ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg
     4621 gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat
     4681 gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc
     4741 gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg
     4801 aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc
     4861 accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac
     4921 ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac
     4981 ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca
     5041 tcctgtctct tgatcagatc cgaaaatgga tatacaagct cccgggagct ttttgcaaaa
     5101 gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg cagaggcggc
     5161 ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact
     5221 gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt
     5281 gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc cacacctggt
     5341 tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt
     5401 tccacaccct aactgacaca cattccacag aattaattcg cgttaaattt ttgttaaatc
     5461 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     5521 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     5581 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     5641 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     5701 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5761 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5821 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtggggata ccccctagag
     5881 ccccagctgg ttctttccgc ctcagaagcc atagagccca ccgcatcccc agcatgcctg
     5941 ctattgtctt cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa
     6001 tgacacctac tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga
     6061 gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa
     6121 ctagaaggca cagtcgaggc tgatcagcgg gtttaaacgg gccctctaga ctcgagttag
     6181 gcaggggcca cctgagaagt ctcggtctta gacacggtgg tgctggcctc atcgaattca
     6241 ccggtgcgtc caccggtatc gatccatgca gtctgggtcc acggcctgca cagaatgcgt
     6301 ctaaaggccc gccggaaatc ctgattgaaa atggtgtaaa tcacaggatt cagtgagcta
     6361 ttgcaataac caatccagaa aaaaaattga aagagcccat gtggtacttt gcaatgctta
     6421 ggacaaatcg cgcccaagga gtaggagaag aaaaatggga accagcagag aacgaatacg
     6481 ccgatcacga cggcgagcac aaaggtgaag cgcttttctc tggtcagctg tgcccgccgg
     6541 cgccaccatt gccctccaat ggcaccgaca ccccttccga gcagcacttg ccctctgagg
     6601 gtagcgagga ctctggagcc ctgtggctgc tgaagaggag gtgagcatgc agaggctgga
     6661 cttacaggca ctgcttgagg ctcgcattcc tcctcttcct cctcttcttc ttcctcctcc
     6721 tcggcctcgt cctcgggact tgcgccgcac accccctcct tctgtccctg cccagagttt
     6781 ggcagcgcag cccaacttgg aggcagtgcg cgggttccgg tatcttcagg ggtctcgccc
     6841 tcttcctttt caccagtaga tttactgtgg ccattgactt ctcttgctga
//
