LOCUS       sequence-430760-        6881 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 6881)
  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 6881)
  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: CCKBR-DuET
FEATURES             Location/Qualifiers
     source          1..6881
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      219..807
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     708..727
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     961..978
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1095..1116
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1131..1136,1137..1154,1155..1161)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1169..1185
                     /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     1174..1196
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1193..1209
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1193..1209
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1258..1379
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1269..1288)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1323..1342
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1553..2347)
                     /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(1664..1683)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2274..2293
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2414..2743)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2428..2563
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2482..2501)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2728..2748
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2757..3185)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2867..2888)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3079..3098
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3231..3455
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3444..3461
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5051..5070)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5052..5070)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5115..5318)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5144..5164)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5319..5698
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5870..5889
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5964..6068
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6069..6137,6138..6881,1..48)
                     /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(6287..6306)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
       61 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
      121 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
      181 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
      241 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
      301 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
      361 accgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
      421 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
      481 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
      541 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
      601 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
      661 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
      721 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
      781 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
      841 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
      901 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
      961 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     1021 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     1081 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     1141 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     1201 agctatgacc atgattacgc caagctctag ctagaggtcg acggtataca gacatgataa
     1261 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     1321 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg
     1381 gggtgggcga agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc
     1441 cggaaaacga ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt
     1501 gatggcaggt tgggcgtcgc ttggtcggtc atttcgaacc ccagagtccc gctcagaaga
     1561 actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa
     1621 gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca
     1681 acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa
     1741 agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat
     1801 cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct
     1861 gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc
     1921 gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca
     1981 gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca
     2041 ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa
     2101 cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct
     2161 cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc
     2221 cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt
     2281 catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt
     2341 caatcatgcg aaacgatcct catcctgtct cttgatcaga tccgaaaatg gatatacaag
     2401 ctcccgggag ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga
     2461 atagctcaga ggcagaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat
     2521 ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg
     2581 ggactatggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     2641 ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc
     2701 ctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac agaattaatt
     2761 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     2821 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     2881 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     2941 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     3001 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     3061 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     3121 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     3181 cgcgtgggga taccccctag agccccagct ggttctttcc gcctcagaag ccatagagcc
     3241 caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc
     3301 caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt
     3361 ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg
     3421 gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc gggtttaaac
     3481 gggccctcta gactcgagtt aggcaggggc cacctgagaa gtctcggtct tagacacggt
     3541 ggtgctggcc tcatcgaatt caccggtgcg tccaccggta tcgatgccgg gccccagggt
     3601 ggaaattgtg gtgtaagaca accgactcag gctagctatg ctcggtgtag gtgggtcctc
     3661 atctggcagt gctcttggcc tggcccgggg agggcgtggg caacaccttg cacaggtttc
     3721 cagacaagcc tgcctaaatc ttctatgcat aaaacaatag acgaggggat tgacacaagc
     3781 actagcatat gacaacagat ggataaagct aattggagct ccactcaggg ccctatgtgc
     3841 cccagggcca tcaaaagctc tccaggtgtt agcactgtat actggcagcc agcagaggaa
     3901 gaacagcacg acgatcacca agagcatcct caccacgcgc ttcttagcca gcagcttcgc
     3961 ttgggtaggt cgacttcctg aacctggccc aggagctgtc agggcggtaa gctccagagc
     4021 tggtctggat ctgggcagct gcacgtagca gccatcagaa tcctcgccca ccgcacctgt
     4081 ttctgggcga catcttccgt tctggtgcac ggcgccggga agaccgcctt ggttccggac
     4141 gcgagactgt gagtcggagt cgctgtcccc gtcgaagcga agtcccaggt aaagttctcg
     4201 gcttatcagt ccgtatgcca ctgccatcac cacccccgga ataaagaaca gcaacagcag
     4261 caagaggacg ctccatgtct gtcttactct agcagaaggc cacctatgga cacattgcaa
     4321 gacccggggg cccactggtt ggacaacagt atacacggga tagggcacca tcaagaggcc
     4381 agagagcagc catgttgcaa ctatgaccct ggctgcgtga gaccgtgttt gccacacccg
     4441 cgcttgcagg ggcctgcaga ttgcggaata tctctccaat gctatggcca ccagggacaa
     4501 ggtgctcacg gacacagaga cccccatcag gtagctaacc gccttgcata tgacggttcc
     4561 aaaaatgaat gttcccataa gattgggaag gagggtaaaa ggcatgcatg ccactgccag
     4621 cagcagatct gaaacagcga gactcagaag gaaagcattg gtcacagttc gcagcctgcg
     4681 gctcagcccc agcaccacaa tgatcagcat attgccgcca acgctcatca gaaagatgac
     4741 ggcgtacaga gttatcctta tcgccagctc caactccctg gttcctgctc ctcgtatgcg
     4801 cggtggctcg caagacagat tgcccacaga ggaggaattc aggaggggtg ccccgggccg
     4861 acaaagggaa gctcctggtc cggggccagt accctgaaca gatcggttaa gtttcaacag
     4921 ctccatatcg atgctggcgt catcatcgtc cttgtagtcg gcgaatacca ggcagaagat
     4981 gtagctcagg gcgatgatcg tcttcatggt ggcgctaagc ttaagtttaa acgctagcca
     5041 gcttgggtct ccctatagtg agtcgtatta atttcgataa gccagtaagc agtgggttct
     5101 ctagttagcc agagagctct gcttatatag acctcccacc gtacacgcct accgcccatt
     5161 tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa
     5221 aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc
     5281 tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat
     5341 acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag
     5401 gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga
     5461 tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc
     5521 cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg
     5581 ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg
     5641 ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa tcaatgtcaa
     5701 cgcgtatatc tggcccgtac atcgcgaagc agcgcaaaac gcctaaccct aagcagattc
     5761 ttcatgcaaa tgtcggtcaa gccttgcctt gttgtagctt aaattttgct cgcgcactac
     5821 tcagcgacct ccaacacaca agcagggagc agatactggc ttaactatgc ggcatcagag
     5881 cagattgtac tgagagtgca ccatagggga tcgggagatc tcccgatccg tcgacgtcag
     5941 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     6001 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     6061 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     6121 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     6181 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     6241 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     6301 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     6361 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     6421 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     6481 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     6541 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6601 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6661 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6721 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6781 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6841 ttatctacac gacggggagt caggcaacta tggatgaacg a
//
