LOCUS       sequence-430835-        6635 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 6635)
  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 6635)
  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: GPR68-DuET
FEATURES             Location/Qualifiers
     source          1..6635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      313..901
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     802..821
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1055..1072
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1189..1210
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1225..1230,1231..1248,1249..1255)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1263..1279
                     /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     1268..1290
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1287..1303
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1287..1303
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1352..1473
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1363..1382)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1417..1436
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1647..2441)
                     /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(1758..1777)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2368..2387
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2508..2837)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2522..2657
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2576..2595)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2822..2842
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2851..3279)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2961..2982)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3173..3192
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3325..3549
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3538..3555
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4899..4918)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4900..4918)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4963..5166)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4992..5012)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5167..5546
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5718..5737
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5812..5916
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5917..5985,5986..6635,1..142)
                     /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(6135..6154)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
       61 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
      121 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
      181 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
      241 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
      301 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
      361 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
      421 ggtaactggc ttcagcagag cgcagatacc aaataccgtt cttctagtgt agccgtagtt
      481 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
      541 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
      601 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
      661 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
      721 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
      781 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
      841 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
      901 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
      961 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     1021 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     1081 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     1141 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     1201 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     1261 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     1321 ctagctagag gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc
     1381 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     1441 tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag
     1501 atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct
     1561 ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc
     1621 ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
     1681 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     1741 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     1801 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     1861 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     1921 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     1981 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     2041 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     2101 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     2161 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     2221 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     2281 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     2341 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     2401 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     2461 gtctcttgat cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct
     2521 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg
     2581 gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
     2641 ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga
     2701 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct
     2761 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     2821 caccctaact gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct
     2881 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     2941 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     3001 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     3061 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     3121 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     3181 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     3241 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc
     3301 agctggttct ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat
     3361 tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac
     3421 acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg
     3481 caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag
     3541 aaggcacagt cgaggctgat cagcgggttt aaacgggccc tctagactcg agttaggcag
     3601 gggccacctg agaagtctcg gtcttagaca cggtggtgct ggcctcatcg aattcaccgg
     3661 tgcgtccacc ggtatcgatt gcaagtctcc cggttggaaa gccgccggac ccgggggagt
     3721 tgggggtttg aaaagcgggg tggagcttgg taagcagttc cggctcctct ccttgcgcgc
     3781 ctgacttacc agaagcctct ggggcaccca gcggatatgc ttcgcgtgcc ctgccggtcc
     3841 ttgaacatgt aaggaaggcc aggcaagctc cgcggaggcg tgcaagatcc ctatgagttg
     3901 tttcggaaac gaagcaatag agcacaggat cggcgacaca attgaaagat gtcagaagga
     3961 gactgaaatg ataggcgtta aagactccct tggcgaagtc acaagatgcc tcccacacgc
     4021 ttctgaccag cagcaacaca tgatatggca ggaagcacgc gaggaaaatg accacagtgc
     4081 tgagcacaag tctctgaatt tggtctttgc gagatttctg agtaccatgg ctgcggcgaa
     4141 cggctcgcag gattccctgg taggaggcca gcagcagaca gataggaaac aggaacccca
     4201 cgagaaatct gtagtaattg atagctcgct gccaagcttg aatagggtag tgctcgaagc
     4261 agaccctgtg ctgattctcg tcctcgatca cttcctcgtg catcaagaag tatattgagg
     4321 tcaggagttc tttagcccag atcaccacgc taacccccac tgcagccttg agtgtccgga
     4381 actggtggaa tcggaaagga tgggctacgg ccaggtagcg gtccacgctg atgcagcaaa
     4441 ggaatcccac ggaaatgtag atattctcat aaagaagaat cccgcacact tggcagctca
     4501 ggtcaccatg ggaccaatta tcatgctgga ggacgtactg cagccaaaag ggaagggagc
     4561 atatgtagaa caggtctgcc actgtcaggt tgcacaagta cactcccaac tcgttgcgag
     4621 cttttatctg caggtaccca aagtagagac tgaggcagtt ggcagggaag cctacgacaa
     4681 gtaccgtaac gtaaaccacg ggagcgaggg tctggtggat agtgtggtcg atggtacagc
     4741 tcatggagct attgtcggca gtgatgttcc ccatatcgat gctggcgtca tcatcgtcct
     4801 tgtagtcggc gaataccagg cagaagatgt agctcagggc gatgatcgtc ttcatggtgg
     4861 cgctaagctt aagtttaaac gctagccagc ttgggtctcc ctatagtgag tcgtattaat
     4921 ttcgataagc cagtaagcag tgggttctct agttagccag agagctctgc ttatatagac
     4981 ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt
     5041 tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag
     5101 acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg
     5161 catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca
     5221 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     5281 gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     5341 ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat
     5401 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     5461 ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta
     5521 ttaataacta gtcaataatc aatgtcaacg cgtatatctg gcccgtacat cgcgaagcag
     5581 cgcaaaacgc ctaaccctaa gcagattctt catgcaaatg tcggtcaagc cttgccttgt
     5641 tgtagcttaa attttgctcg cgcactactc agcgacctcc aacacacaag cagggagcag
     5701 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc ataggggatc
     5761 gggagatctc ccgatccgtc gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5821 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5881 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5941 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     6001 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     6061 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     6121 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     6181 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6241 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6301 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6361 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6421 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6481 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6541 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6601 attgctgata aatctggagc cggtgagcgt gggtc
//
