LOCUS       sequence-430731-        6692 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 6692)
  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 6692)
  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: S1PR4-DuET
FEATURES             Location/Qualifiers
     source          1..6692
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(609..628)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(610..628)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(673..876)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(702..722)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        877..1256
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1428..1447
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1522..1626
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1627..1695,1696..2487)
                     /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(1845..1864)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2658..3246
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3147..3166
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3400..3417
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3534..3555
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3570..3575,3576..3593,3594..3600)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3608..3624
                     /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     3613..3635
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3632..3648
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3632..3648
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3697..3818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3708..3727)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3762..3781
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3992..4786)
                     /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(4103..4122)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4713..4732
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4853..5182)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4867..5002
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4921..4940)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5167..5187
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5196..5624)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5306..5327)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5518..5537
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5670..5894
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5883..5900
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 ctcgactggt ctttgttgcg ccggactcag cgacagggcg caccatcgta gcaaaccgct
       61 cccctgcggt gaacagcaag ctgaaagtgg aagccgccag ggctgtgaac agcaggccct
      121 cccggaggaa ccattgggca ggagcaagcc ggaaggtgcg ggcgccagac aggagcacat
      181 tggcaagata ggcggcccct gtcaggaggt cactgagggt aatgttcacg aggcagtagt
      241 atacccagcg tcgggagcgc atatggctag tgatggcggc gagcacaagg aggttttcca
      301 aaaccaccag gcagctagct gcgacggaaa ggcccctcag agcaccgagg cccccgtctt
      361 cagggccacc gcgccctgcc agtcggccgg aatgattgta gtgcaggacg atcagcctac
      421 tgtggccccc ggcggcaagt tgctgacagc tctcaggggc aacgggggtt ccggtagcgt
      481 tcatatcgat gctggcgtca tcatcgtcct tgtagtcggc gaataccagg cagaagatgt
      541 agctcagggc gatgatcgtc ttcatggtgg cgctaagctt aagtttaaac gctagccagc
      601 ttgggtctcc ctatagtgag tcgtattaat ttcgataagc cagtaagcag tgggttctct
      661 agttagccag agagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg
      721 cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa
      781 caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta
      841 tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac
      901 gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc
      961 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     1021 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc
     1081 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     1141 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct
     1201 atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcaacg
     1261 cgtatatctg gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt
     1321 catgcaaatg tcggtcaagc cttgccttgt tgtagcttaa attttgctcg cgcactactc
     1381 agcgacctcc aacacacaag cagggagcag atactggctt aactatgcgg catcagagca
     1441 gattgtactg agagtgcacc ataggggatc gggagatctc ccgatccgtc gacgtcaggt
     1501 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     1561 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     1621 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     1681 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     1741 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     1801 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     1861 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     1921 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     1981 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     2041 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     2101 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     2161 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     2221 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     2281 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     2341 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     2401 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     2461 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     2521 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     2581 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2641 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2701 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2761 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ccgttcttct agtgtagccg
     2821 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2881 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2941 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3001 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3061 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3121 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3181 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3241 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3301 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3361 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3421 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     3481 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     3541 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     3601 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     3661 aagctctagc tagaggtcga cggtatacag acatgataag atacattgat gagtttggac
     3721 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     3781 ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa gaactccagc
     3841 atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat tccgaagccc
     3901 aacctttcat agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt gggcgtcgct
     3961 tggtcggtca tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga
     4021 aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc
     4081 attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt
     4141 ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga
     4201 tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg
     4261 ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat
     4321 cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt
     4381 ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca
     4441 tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt
     4501 cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag
     4561 gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg
     4621 caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca
     4681 cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca
     4741 cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga aacgatcctc
     4801 atcctgtctc ttgatcagat ccgaaaatgg atatacaagc tcccgggagc tttttgcaaa
     4861 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gcagaggcgg
     4921 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
     4981 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat
     5041 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg
     5101 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     5161 ttccacaccc taactgacac acattccaca gaattaattc gcgttaaatt tttgttaaat
     5221 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     5281 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     5341 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     5401 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     5461 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     5521 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     5581 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtggggat accccctaga
     5641 gccccagctg gttctttccg cctcagaagc catagagccc accgcatccc cagcatgcct
     5701 gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc ccagaataga
     5761 atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa ggacagtggg
     5821 agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag atggctggca
     5881 actagaaggc acagtcgagg ctgatcagcg ggtttaaacg ggccctctag actcgagtta
     5941 ggcaggggcc acctgagaag tctcggtctt agacacggtg gtgctggcct catcgaattc
     6001 accggtgcgt ccaccggtat cgataattga gcgaacggag cttatactag acaaaggttc
     6061 tctcattcta aagctcaagg accgactgcc tcgaaaagaa tccctgggcc tgagggaact
     6121 atctgtggtt gatgcgccac tatgtgcttc tacggctctg gccagacaat cccctggccc
     6181 tctcattcca aggcgcagac agccgcaaca caggaagcta agtacggccc tgcacacctc
     6241 cctacttctg aatgagtaga tgattggatt cactgcgctg ttgagcacgg cgagggccag
     6301 aatccagtcc atcccgcgca gatactcttg cgcccagagg tttgagccga aaacgtctgc
     6361 cagcaacagc ccaaacaggg ggccccagca tacgaggaag gcaagcagaa tcatcagcac
     6421 tgttttcaag aggcgccggg ctttcctccg ggcggcgggg cggggtgctt tctgaccgga
     6481 ggcctgcaca agcctaaaaa tagcaccata cagtcccata attgtggcca acacaccggc
     6541 gaagatcacg aggcaaaaca gaatatatct cttactgtaa agtggcagga ggctgctgca
     6601 cctatcaaag gcacacaaac agttccaccc aagaagtggc agcatgccca gaagtgccgc
     6661 gagcaaccaa cagagtccga tgaatccgta aa
//
