LOCUS       sequence-430887-        6650 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 6650)
  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 6650)
  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: PRRP-DuET
FEATURES             Location/Qualifiers
     source          1..6650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(588..607)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(589..607)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(652..855)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(681..701)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        856..1235
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1407..1426
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1501..1605
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1606..1674,1675..2466)
                     /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(1824..1843)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2637..3225
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3126..3145
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3379..3396
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3513..3534
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3549..3554,3555..3572,3573..3579)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3587..3603
                     /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     3592..3614
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3611..3627
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3611..3627
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3676..3797
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3687..3706)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3741..3760
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3971..4765)
                     /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(4082..4101)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4692..4711
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4832..5161)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4846..4981
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4900..4919)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5146..5166
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5175..5603)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5285..5306)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5497..5516
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5649..5873
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5862..5879
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 tcagggtgaa cacactgaca tagactgtaa ctggctgcaa gaagaacacc aagtggcaca
       61 gtccaccccc gaacacccac cctctgggct caaaggcgta tgccagggtg aggggcacac
      121 aagccgtaca cataagcaca tcgctcaggg cgagatttcc aatcaaaaaa ttagtcacgt
      181 tgtgcagtct cctaacccta gcaataacca ggaccaacag acagttgcca accaggccga
      241 ccacgaccac cacggagtag agcaggacta tcaggccttt gagctgatgc accagctgca
      301 gactctggaa tggggtcact gcgggagcat cggcccccgc tactgatcca tttccggcgg
      361 acgcctcggc gctttggttc gctggtgttg tgactgccgg cgggagtcct gaaaagaggt
      421 cactgaccct tggcccgcgt gtggtgcttg aggccatatc gatgctggcg tcatcatcgt
      481 ccttgtagtc ggcgaatacc aggcagaaga tgtagctcag ggcgatgatc gtcttcatgg
      541 tggcgcggcc gctaagctta agtttaaacg ctagccagct tgggtctccc tatagtgagt
      601 cgtattaatt tcgataagcc agtaagcagt gggttctcta gttagccaga gagctctgct
      661 tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg cggagttgtt
      721 acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat tgacgtcaat
      781 ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat tgatgtactg
      841 ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg
      901 aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac
      961 gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta
     1021 ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac
     1081 atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt
     1141 taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat
     1201 tgattactat taataactag tcaataatca atgtcaacgc gtatatctgg cccgtacatc
     1261 gcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaaatgt cggtcaagcc
     1321 ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca acacacaagc
     1381 agggagcaga tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     1441 taggggatcg ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg
     1501 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     1561 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     1621 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     1681 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     1741 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     1801 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     1861 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     1921 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     1981 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2041 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2101 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     2161 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     2221 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     2281 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     2341 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2401 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2461 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     2521 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     2581 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2641 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2701 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2761 agagcgcaga taccaaatac cgttcttcta gtgtagccgt agttaggcca ccacttcaag
     2821 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2881 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2941 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3001 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3061 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3121 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3181 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3241 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3301 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3361 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     3421 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     3481 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     3541 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     3601 caatttcaca caggaaacag ctatgaccat gattacgcca agctctagct agaggtcgac
     3661 ggtatacaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
     3721 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     3781 gctgcaataa acaagttggg gtgggcgaag aactccagca tgagatcccc gcgctggagg
     3841 atcatccagc cggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg
     3901 gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc
     3961 agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg
     4021 gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag
     4081 caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac
     4141 agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc
     4201 catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt
     4261 cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt
     4321 ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag
     4381 ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag
     4441 gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc
     4501 ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc
     4561 acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga
     4621 caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga
     4681 ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg
     4741 cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc
     4801 cgaaaatgga tatacaagct cccgggagct ttttgcaaaa gcctaggcct ccaaaaaagc
     4861 ctcctcacta cttctggaat agctcagagg cagaggcggc ctcggcctct gcataaataa
     4921 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
     4981 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac
     5041 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca
     5101 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca
     5161 cattccacag aattaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     5221 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     5281 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     5341 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     5401 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     5461 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     5521 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     5581 taatgcgccg ctacagggcg cgtggggata ccccctagag ccccagctgg ttctttccgc
     5641 ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc
     5701 ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg
     5761 cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa
     5821 ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc
     5881 tgatcagcgg gtttaaacgg gccctctaga ttaggcaggg gccacctgag aagtctcggt
     5941 cttagacacg gtggtgctgg cctcatcgaa ttcaccggtg cgtccaccgg tatcgataat
     6001 taccacgctt acggtcatat tctgcccatg tggagcgatt tttctaggcc aagcgacgag
     6061 cagttttctc agctcttccc taaagctgtc atggagccag gcgtaaatga atggattata
     6121 gcaagcactt gacatcgcca gccaatggca gagcagctga accagaccga atgcgtatgg
     6181 gtctatcgca tgcgggtcca gatcccgcag aaggttaaaa acgtgcagcg gcagccagca
     6241 cacagcaaag accaccacca ccacgacaag gagacaaaag gttcgtcttc gtcgtgcccg
     6301 gtcccaatca gcctgagact gagtgacaca gccaggaacg actcgatttc taagcttaac
     6361 gctcactcgt acatagctca ggaggataac cagaagagga aggaggtagg tcacaagcaa
     6421 aagtccccag gcataaagct gtctctgccg ctcctgggac ccccaaaact cttcgcagag
     6481 gcgcacgtcg tggggcttca gctcaacgtg ataagtgtgg accgcagcag gcagcgccag
     6541 cactgcgctc aaggcccaaa tggccaggac ggcataggcg ctcagtcgga gtgagattcg
     6601 ccgtctcagg ggatgcacca gcaccacgta tcggtctaca gcgatggtgg
//
