LOCUS       sequence-431750-        6794 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 6794)
  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 6794)
  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: AVPR1A-DuET
FEATURES             Location/Qualifiers
     source          1..6794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      303..891
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     792..811
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1045..1062
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1179..1200
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1215..1220,1221..1238,1239..1245)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1253..1269
                     /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     1258..1280
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1277..1293
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1277..1293
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1342..1463
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1353..1372)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1407..1426
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1637..2431)
                     /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(1748..1767)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2358..2377
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2498..2827)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2512..2647
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2566..2585)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2812..2832
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2841..3269)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2951..2972)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3163..3182
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3315..3539
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3528..3545
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5048..5067)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5049..5067)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5112..5315)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5141..5161)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5316..5695
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5867..5886
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5961..6065
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6066..6134,6135..6794,1..132)
                     /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(6284..6303)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
       61 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
      121 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
      181 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
      241 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
      301 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
      361 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
      421 ttcagcagag cgcagatacc aaataccgtt cttctagtgt agccgtagtt aggccaccac
      481 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
      541 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
      601 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
      661 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
      721 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      781 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      841 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      901 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      961 gcgttatccc ctgattctgt agataaccgt attaccgcct ttgagtgagc tgataccgct
     1021 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     1081 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     1141 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     1201 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     1261 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct ctagctagag
     1321 gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
     1381 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     1441 ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag atccccgcgc
     1501 tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct ttcatagaag
     1561 gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc ggtcatttcg
     1621 aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg
     1681 aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct
     1741 cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc
     1801 ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg
     1861 catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga
     1921 acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac
     1981 cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc
     2041 aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct
     2101 cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc
     2161 agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg
     2221 ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg
     2281 tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc
     2341 agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag
     2401 aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat
     2461 cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct aggcctccaa
     2521 aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg gcctctgcat
     2581 aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg
     2641 gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt
     2701 gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga
     2761 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact
     2821 gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct cattttttaa
     2881 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     2941 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     3001 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     3061 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3121 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3181 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3241 cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc agctggttct
     3301 ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat tgtcttccca
     3361 atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac acctactcag
     3421 acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg caccttccag
     3481 ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag aaggcacagt
     3541 cgaggctgat cagcgggttt aaacgggccc tctagattag gcaggggcca cctgagaagt
     3601 ctcggtctta gacacggtgg tgctggcctc atcgaattca ccggtgcgtc caccggtatc
     3661 gatggtggac acggggatga atttaatgct tttggagctc tttggactgt ccttccacat
     3721 ccctgtagag tttgtggggg atctattatt agagtaaaat gtctgtctcc gactcatgga
     3781 gtcagtgtcc tctttattaa acttttcctt catgttctgg cagcatggaa aggactggac
     3841 gcaatcctgc agaaggtgcc cggaaaagaa catgtagatc caggggttac agcatgagtt
     3901 gagggacccc agcagggccg tgattgtaat ggtggggttt tcagactcgg tccacacgct
     3961 catcggatcc cacacggacc acatttggat tatgaaaaaa ggggcccagc agacaatata
     4021 tgctgtaacg atgacgaatg tcattttgac ggtgcgaatc tttgcccgag agatagattt
     4081 cacggaagat acgcacggag ccaacagaaa gcctttctga aatgccacac cggcctgctc
     4141 agcaccctta ctctgtcggg atgcagtctt gccgcgtaca ttgcaccaga tgttgtagca
     4201 aatgaagccg taacatgtcc caagtatcac cactggcgcg acaaatattc cgcccgtcat
     4261 ccacgtcaca tacgcgcgag atccccatgg ctggataaat gttgcccaac aatcgcgtgc
     4321 tttagtcaca ttgttcactt caatcataga aaagacgaaa tactgggggg ttgaaagcac
     4381 aaaagagagc acccaagctg cggctatcat aagccgagat ctgcgggcag gctgctgcag
     4441 tgtcttcagg ggatggcaca cggcaatgta gcggtcggcc gtcatgacca cgagcatata
     4501 ggcgctggcg aacatcccga agacctgaag atgcttcacc actctgcaca accagtctgg
     4561 gcccctaaag cgatatgtga tgtcccagca catctggggc agtacttgaa aaaaagcgac
     4621 tgccagatcg gccaaagaca gatgcctaat gaacaaatgc attctgctcg tcttgcgtgg
     4681 ggttctatgc agagccaaga ggacgctgct attgccgagg acagccactg cgaatgtgac
     4741 ggccagcacg gcgatctcca gtttagccag ctcctcgttg cgtacgtccc ttggggggcc
     4801 gttgccctcg cccagagcct cggcttctct ggatgtattt ccggctccgg tggcaagggg
     4861 ccaccaagga ctgctgttgc cactgggccc tgcgtcagga ccagcactca acctcatatc
     4921 gatgctggcg tcatcatcgt ccttgtagtc ggcgaatacc aggcagaaga tgtagctcag
     4981 ggcgatgatc gtcttcatgg tggcgcggcc gctaagctta agtttaaacg ctagccagct
     5041 tgggtctccc tatagtgagt cgtattaatt tcgataagcc agtaagcagt gggttctcta
     5101 gttagccaga gagctctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc
     5161 gtcaatgggg cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac
     5221 aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat
     5281 ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg
     5341 tagatgtact gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg
     5401 ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt
     5461 actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct
     5521 attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc
     5581 ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta
     5641 tgaactaatg accccgtaat tgattactat taataactag tcaataatca atgtcaacgc
     5701 gtatatctgg cccgtacatc gcgaagcagc gcaaaacgcc taaccctaag cagattcttc
     5761 atgcaaatgt cggtcaagcc ttgccttgtt gtagcttaaa ttttgctcgc gcactactca
     5821 gcgacctcca acacacaagc agggagcaga tactggctta actatgcggc atcagagcag
     5881 attgtactga gagtgcacca taggggatcg ggagatctcc cgatccgtcg acgtcaggtg
     5941 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     6001 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     6061 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     6121 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     6181 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     6241 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     6301 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     6361 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     6421 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6481 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6541 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6601 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6661 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6721 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6781 ggtctcgcgg tatc
//
