LOCUS       sequence-430896-        7136 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 7136)
  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 7136)
  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: EDNRB-DuET
FEATURES             Location/Qualifiers
     source          1..7136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      481..1069
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     970..989
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1223..1240
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1357..1378
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1393..1398,1399..1416,1417..1423)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1431..1447
                     /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     1436..1458
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1455..1471
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1455..1471
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1520..1641
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1531..1550)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1585..1604
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1815..2609)
                     /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(1926..1945)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2536..2555
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2676..3005)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2690..2825
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2744..2763)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2990..3010
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3019..3447)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3129..3150)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3341..3360
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3493..3717
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3706..3723
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5568..5587)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5569..5587)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5632..5835)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5661..5681)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5836..6215
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6387..6406
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6481..6585
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6586..6654,6655..7136,1..310)
                     /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(6804..6823)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
       61 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
      121 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
      181 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
      241 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
      301 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
      361 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
      421 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
      481 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
      541 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      601 cagcagagcg cagataccaa ataccgttct tctagtgtag ccgtagttag gccaccactt
      661 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      721 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      781 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      841 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      901 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      961 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     1021 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     1081 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     1141 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     1201 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     1261 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     1321 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     1381 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     1441 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1501 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1561 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1621 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1681 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1741 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     1801 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     1861 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     1921 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     1981 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     2041 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     2101 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     2161 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     2221 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     2281 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     2341 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     2401 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     2461 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     2521 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     2581 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     2641 gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag gcctccaaaa
     2701 aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc ctctgcataa
     2761 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     2821 gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc
     2881 atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga
     2941 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga
     3001 cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     3061 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     3121 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     3181 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     3241 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     3301 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     3361 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     3421 cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag ctggttcttt
     3481 ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat
     3541 cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac
     3601 aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg
     3661 tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg
     3721 aggctgatca gcgggtttaa acgggccctc tagattaggc aggggccacc tgagaagtct
     3781 cggtcttaga cacggtggtg ctggcctcat cgaattcacc ggtgcgtcca ccggtatcga
     3841 tgctagatga gtatttattg gaggaccgaa agttatcata gccgtggtca tttgctttga
     3901 acttcagaca gctctgtttt tcctccaaac tctgcttctc ttcaaaagat tggcaccaac
     3961 agcacagaca gctcttgaag caattcttaa atctcttgct cacgagatag agtgcaatgg
     4021 gattaataca actgttcagg gaggccatgt taatgccgat ataatcgagc acgaggagga
     4081 agctgagcag ctcgcatctg ttaggatcat tctgattgta cagagtcagc ttgaggattc
     4141 tggaaaggtg cagaggcagc caacagagag caaagaccag gaccaagcaa aacacggtct
     4201 tagccacctc ccgtcgttgt ttcaggtggt cattgagagc aatctgcata ccggattttt
     4261 tccgaagcat ctcgcaagtc ataagggtgt agaagaaagc agttattgcc aatgggaggc
     4321 agaagtaaaa agagaagagc caccaatctt tagcagtctt atagaactgc atgaacgctg
     4381 tcttttgcac agggtgcagg agacagatcc ggaggtaaga tcccttgtag tccattgtga
     4441 ttatatcaaa cccgatggcc tcaggcacag cgagaacgac actcaccacc cagatgagca
     4501 cgatctcaac ggcagtccac ttgggaacac cgatcccctt gatgcgtgac cagctggcaa
     4561 cagctcggta tctatcaatg gacagggcgc agagggacag cactgtgatc cccacggagg
     4621 ccttctggat gaagggaacc agtttgcaca tttctgcgcc aaagggccag tcctcggcaa
     4681 ggagcttgta cacattgata ggaatgtcga tgacgatatg caggagatcg cccagcgcca
     4741 gactggcgat caggatgttg ggtccgttgc gcatgcactt atttttgtat atgattctga
     4801 gcaacgtgga gtttcctatt atgcccagta caaagaccag gcaagacact acggtgttta
     4861 tatacttgaa ggtctctttg atctctatag gtccctgaca gggcgggggg gaaatagttc
     4921 ttggtgggct gccggctgtt ctatcgcctt ttggtacttc ggcgggagcc agagacctag
     4981 ccaatgatgc gttagatccc ttgggccaaa gtgttttggt gggaggggtc attatctcag
     5041 cagtctgcag caagggtgta gcgcggtcgg gtggaaaacc tctttcttcg ccccatatgc
     5101 gactcagacc gcaagccaaa acgagagcta ccaaagctcg gccgcagaga ctaggtggcg
     5161 gctgcattga agagccagct ccactaggcg tcctatgacc gcttctaagc ttaagagcct
     5221 ctcttatttg acagacgagg tgcaaggcca cgaatgggga ccaccggccc ggcagtctaa
     5281 agccggcagg gttccatccc gcccgtgggc tagaacaggc ttctcttgaa gagcatgctg
     5341 gacccgccct gacaaaccgc tgtgaaaatg cgaggcaatg aagtctccac cgtttggacg
     5401 gagtctcagc cgccatcagg caggtagact tgttcatatc gatgctggcg tcatcatcgt
     5461 ccttgtagtc ggcgaatacc aggcagaaga tgtagctcag ggcgatgatc gtcttcatgg
     5521 tggcgcggcc gctaagctta agtttaaacg ctagccagct tgggtctccc tatagtgagt
     5581 cgtattaatt tcgataagcc agtaagcagt gggttctcta gttagccaga gagctctgct
     5641 tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg cggagttgtt
     5701 acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat tgacgtcaat
     5761 ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat tgatgtactg
     5821 ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg
     5881 aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac
     5941 gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta
     6001 ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac
     6061 atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt
     6121 taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat
     6181 tgattactat taataactag tcaataatca atgtcaacgc gtatatctgg cccgtacatc
     6241 gcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaaatgt cggtcaagcc
     6301 ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca acacacaagc
     6361 agggagcaga tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     6421 taggggatcg ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg
     6481 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     6541 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     6601 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     6661 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     6721 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     6781 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     6841 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6901 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6961 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     7021 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     7081 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaat
//
