LOCUS       sequence-431748-        6629 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 6629)
  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 6629)
  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: AGTR2-DuET
FEATURES             Location/Qualifiers
     source          1..6629
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     422..441
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     675..692
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    809..830
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(845..850,851..868,869..875)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    883..899
                     /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     888..910
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     907..923
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     907..923
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    972..1093
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(983..1002)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1037..1056
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1267..2061)
                     /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(1378..1397)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1988..2007
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2128..2457)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2142..2277
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2196..2215)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2442..2462
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2471..2899)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2581..2602)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2793..2812
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2945..3169
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3158..3175
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4513..4532)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4514..4532)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4577..4780)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4606..4626)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4781..5160
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5332..5351
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5426..5530
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5531..5599,5600..6391)
                     /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(5749..5768)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6562..6629,1..521)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
       61 cgcagatacc aaataccgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
      121 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
      181 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
      241 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
      301 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
      361 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
      421 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
      481 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
      541 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
      601 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
      661 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
      721 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
      781 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
      841 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
      901 ttcacacagg aaacagctat gaccatgatt acgccaagct ctagctagag gtcgacggta
      961 tacagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     1021 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     1081 caataaacaa gttggggtgg gcgaagaact ccagcatgag atccccgcgc tggaggatca
     1141 tccagccggc gtcccggaaa acgattccga agcccaacct ttcatagaag gcggcggtgg
     1201 aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc ggtcatttcg aaccccagag
     1261 tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc
     1321 ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat
     1381 atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc
     1441 gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg
     1501 ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc
     1561 tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat
     1621 ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg
     1681 atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc
     1741 aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc
     1801 cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga
     1861 tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa
     1921 aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt
     1981 ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg
     2041 caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cagatccgaa
     2101 aatggatata caagctcccg ggagcttttt gcaaaagcct aggcctccaa aaaagcctcc
     2161 tcactacttc tggaatagct cagaggcaga ggcggcctcg gcctctgcat aaataaaaaa
     2221 aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg
     2281 cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct
     2341 gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct
     2401 ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt
     2461 ccacagaatt aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     2521 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     2581 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     2641 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     2701 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     2761 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     2821 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     2881 gcgccgctac agggcgcgtg gggatacccc ctagagcccc agctggttct ttccgcctca
     2941 gaagccatag agcccaccgc atccccagca tgcctgctat tgtcttccca atcctccccc
     3001 ttgctgtcct gccccacccc accccccaga atagaatgac acctactcag acaatgcgat
     3061 gcaatttcct cattttatta ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa
     3121 ggcacggggg aggggcaaac aacagatggc tggcaactag aaggcacagt cgaggctgat
     3181 cagcgggttt aaacgggccc tctagattag gcaggggcca cctgagaagt ctcggtctta
     3241 gacacggtgg tgctggcctc atcgaattca ccggtgcgtc caccggtatc gatggacacg
     3301 aaagtttcca tttcccgaag ggaagaggat tttcgacaag acatggactc gcgtttgccc
     3361 tgcaaccacg tgataggcac ccggaacacg gatcgcaatt tttgctgaaa tctattccca
     3421 acaaaacaat acaagaacgg gttaacgcaa ctattagtaa agccaaggag gatggcgaaa
     3481 ggcagggcga ggtcaatcac ggcaatcacc tcacaagagt taatgacgcc catccaagcc
     3541 agcgcgtcca gaaatgtcag cacatggaaa ggaagccagc agataataaa agccagcaca
     3601 accgcggcag ccattttcag cacctgatcg cgggtaatcc tgttcttccc ataggaatta
     3661 gtcttgagca ggtgctttct tatgccgaag tagcacgtgg ctataaaaat gagggggatg
     3721 ataaatccca gaatattctt catcagggct atgcccgcac tccactgcgc gtacttctct
     3781 ggtggaaacg ccataataca tgcgttcacc cccaaatact cgattgtcct aacgtcccgg
     3841 aagtaaaagg taggaaggga tgacaggcag gccatacacc agaccagtgg cacaatgtaa
     3901 gaggcctgcc aagggtttct tctctgactc aggaatgggt agatcacaga ctggtatctg
     3961 tccacactca tacaggtaat aaaaaaaatg ctagcaaaca tattcagggt caggaaggag
     4021 ccaaacactt tgcacataac tggtccgaac agccaatcgt agcggtatga gtagtaagtt
     4081 gcccaaaggg ggagggtagc cagcagcagc aggtcggcaa cggccagatt aaaaatgtat
     4141 atgctggaca ctttcttggg gcccttttga cagcaaaaca gggtcaccac gacgatattt
     4201 accagaaatc cgattacaaa gataatgtaa tacaggatgg gaatggcgtc caggtgttta
     4261 tcactcggct tctgactgca attcagagtg gattcattat ttccggagat atttacaagg
     4321 ccaaaatgca ggccacttgt aatgttctta gaggtagtgg ccaaggtgga gtttcccttc
     4381 atatcgatgc tggcgtcatc atcgtccttg tagtcggcga ataccaggca gaagatgtag
     4441 ctcagggcga tgatcgtctt catggtggcg cggccgctaa gcttaagttt aaacgctagc
     4501 cagcttgggt ctccctatag tgagtcgtat taatttcgat aagccagtaa gcagtgggtt
     4561 ctctagttag ccagagagct ctgcttatat agacctccca ccgtacacgc ctaccgccca
     4621 tttgcgtcaa tggggcggag ttgttacgac attttggaaa gtcccgttga ttttggtgcc
     4681 aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt gagtcaaacc
     4741 gctatccacg cccattgatg tactgccaaa accgcatcac catggtaata gcgatgacta
     4801 atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc
     4861 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt
     4921 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
     4981 tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt
     5041 tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg
     5101 ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc
     5161 aacgcgtata tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat
     5221 tcttcatgca aatgtcggtc aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact
     5281 actcagcgac ctccaacaca caagcaggga gcagatactg gcttaactat gcggcatcag
     5341 agcagattgt actgagagtg caccataggg gatcgggaga tctcccgatc cgtcgacgtc
     5401 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5461 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5521 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5581 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5641 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5701 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5761 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5821 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5881 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5941 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6001 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6061 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6121 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6181 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6241 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6301 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6361 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6421 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6481 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6541 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6601 aacaaaaaaa ccaccgctac cagcggtgg
//
