LOCUS       sequence-430730-        6686 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 6686)
  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 6686)
  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: S1PR1-DuET
FEATURES             Location/Qualifiers
     source          1..6686
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(25..42)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    31..255
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      301..729
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(388..407)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     598..619
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(738..758)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        743..1072
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      923..1058
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     985..1004
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1139..1933
                     /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(1193..1212)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1803..1822
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2107..2228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2144..2163)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2198..2217
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2277..2293)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2277..2293)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2290..2312)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2301..2317
                     /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)."
     promoter        complement(join(2325..2331,2332..2349,2350..2355))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2370..2391
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2508..2525)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2679..3267)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2759..2778)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3438..4229,4230..4298))
                     /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     4061..4080
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4299..4403)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4478..4497)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4669..5048
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5049..5252
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5203..5223
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5297..5316
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5297..5315
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
       61 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
      121 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
      181 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
      241 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
      301 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
      361 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      421 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      481 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      541 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      601 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      661 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      721 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
      781 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
      841 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
      901 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
      961 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     1021 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     1081 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     1141 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     1201 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     1261 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     1321 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     1381 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     1441 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     1501 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     1561 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     1621 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     1681 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     1741 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     1801 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     1861 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     1921 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     1981 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     2041 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     2101 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2161 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2221 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     2281 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     2341 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     2401 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     2461 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     2521 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2581 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     2641 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2701 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2761 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2821 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2881 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2941 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3001 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3061 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     3121 acggtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3181 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3241 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3301 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3361 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3421 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3481 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3541 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3601 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3661 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3721 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3781 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3841 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3901 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3961 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4021 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4081 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4141 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4201 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4261 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4321 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4381 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     4441 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     4501 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     4561 aaatttaagc tacaacaagg caaggcttga ccgacatttg catgaagaat ctgcttaggg
     4621 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     4681 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     4741 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     4801 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     4861 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     4921 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     4981 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     5041 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     5101 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     5161 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     5221 gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt
     5281 actggcttat cgaaattaat acgactcact atagggagac ccaagctggc tagcgtttaa
     5341 acttaagctt agcgccacca tgaagacgat catcgccctg agctacatct tctgcctggt
     5401 attcgccgac tacaaggacg atgatgacgc cagcatcgat atgggaccaa catcagtccc
     5461 attggttaaa gcccatcgaa gctccgtgag cgactacgtg aactacgaca tcatcgtgag
     5521 gcactataac tataccggaa agctcaacat atccgccgat aaagagaata gtattaaact
     5581 gacctctgtc gtcttcatac tgatctgttg tttcatcatt ctcgaaaaca ttttcgtgct
     5641 ccttaccatc tggaaaacca aaaagttcca cagacctatg tactatttta tcggaaatct
     5701 ggccctgtct gacttgttgg ccggggtggc ctatactgca aacttgctgc tctcaggcgc
     5761 cactacatac aaactcacgc ccgctcagtg gtttctgagg gagggctcca tgtttgtggc
     5821 gctgagcgct agcgtgttca gcctgctggc tatagccatc gaacgataca ttactatgct
     5881 gaagatgaag ctgcacaatg gctctaacaa ttttaggctc tttctcctta tatccgcgtg
     5941 ctgggtgatt tctctgattc tcggggggct gcccatcatg gggtggaatt gcatctctgc
     6001 tctgtcatca tgctccactg tgcttcccct gtatcataag cactatatat tgttttgtac
     6061 cactgttttc accctcttgc tgctcagcat tgtgattctc tactgccgca tatactcctt
     6121 ggtccggaca agatccaggc gcctgacgtt ccggaaaaac atctctaagg cgtctagatc
     6181 atccgagaaa agcctggcgc tcctgaagac cgtcatcatc gtgttgagcg tctttatcgc
     6241 ttgctgggcc cccctgttta tcttgctcct gctggatgta gggtgtaaag tgaagacgtg
     6301 cgatatcctg ttccgggccg agtattttct tgtcctggca gtcctgaact ccggcactaa
     6361 tcctatcatt tataccctga ccaataaaga aatgcgccgc gccttcatca ggattatgag
     6421 ctgctgtaag tgtccctccg gagactctgc tggcaaattt aagcggccaa tcatagccgg
     6481 catggagttc tccagatcta aatccgacaa tagctcacac cctcagaaag atgaaggcga
     6541 caatcctgaa acaattatga gtagtggtaa tgtgaatagc tcaagcatcg ataccggtgg
     6601 acgcaccggt gaattcgatg aggccagcac caccgtgtct aagaccgaga cttctcaggt
     6661 ggcccctgcc taactcgagt ctagag
//
