LOCUS       sequence-444925-        6617 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 6617)
  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 6617)
  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: PTGFR-DuET
FEATURES             Location/Qualifiers
     source          1..6617
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(49..68)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        240..619
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        620..823
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     774..794
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     868..887
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        868..886
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2213..2230)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2219..2443
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2489..2917
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2576..2595)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2786..2807
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2926..2946)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2931..3260
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3111..3246
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3173..3192
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3327..4121
                     /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(3381..3400)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3991..4010
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4295..4416
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4332..4351)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4386..4405
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4465..4481)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4465..4481)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4478..4500)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4489..4505
                     /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(4513..4519,4520..4537,4538..4543))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4558..4579
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4696..4713)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4867..5455)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4947..4966)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5626..6417,6418..6486))
                     /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     6249..6268
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6487..6591)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
       61 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
      121 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacattt gcatgaagaa
      181 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
      241 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      301 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      361 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      421 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      481 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      541 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      601 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      661 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      721 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      781 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
      841 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg
      901 ctagcgttta aacttaagct tagcggccgc gccaccatga agacgatcat cgccctgagc
      961 tacatcttct gcctggtatt cgccgactac aaggacgatg atgacgccag catcgatatg
     1021 agcatgaaca atagcaagca gcttgtgagt ccagctgctg ccctgcttag caatactacc
     1081 tgccagaccg agaataggct gagcgtgttt ttttccgtga ttttcatgac cgtgggtatt
     1141 ttgagcaaca gccttgcaat cgccattctt atgaaggcat atcaaagatt tcgacaaaag
     1201 tccaaagcct cattccttct cctcgcatca ggtctcgtga ttactgactt cttcggccac
     1261 ctgattaacg gtgctatcgc agtgttcgtt tacgcctcag acaaggaatg gatccgattc
     1321 gaccaatcaa acgtcctctg tagtatcttc ggaatctgta tggtgttttc tgggttgtgc
     1381 ccacttcttc tgggtagcgt gatggcaata gaacggtgca taggggtgac gaagcccatc
     1441 ttccactcca ccaagatcac ctcaaagcac gtgaagatga tgctgtccgg cgtgtgcctc
     1501 ttcgctgttt ttatcgccct gctcccaata ctggggcacc gggactacaa aattcaagcc
     1561 tctaggacat ggtgctttta taacactgag gatatcaaag attgggaaga tcggttctat
     1621 ctgttgttgt tttcattcct gggcctgctg gcattgggcg tttctctcct gtgcaacgcc
     1681 atcaccggca tcaccctgct tcgcgtaaag tttaaatctc agcagcatcg ccaggggcgc
     1741 agtcaccacc tggagatggt tatacagctc ctggccatca tgtgcgtgag ctgtatttgc
     1801 tggagccctt tcttggtcac gatggccaac atcggcatca atggaaacca ttccctcgaa
     1861 acatgtgaaa caacactgtt cgccctgagg atggccactt ggaatcagat actggatcct
     1921 tgggtctaca tcctgctgcg gaaagccgtt ctgaaaaatc tctataagct ggcgagccaa
     1981 tgttgcggag tgcacgtgat cagtctccat atctgggagt tgtctagcat taagaactca
     2041 ctgaaagtcg ccgcgattag cgaatccccc gtggcagaaa aatccgccag taccatcgat
     2101 accggtggac gcaccggtga attcgatgag gccagcacca ccgtgtctaa gaccgagact
     2161 tctcaggtgg cccctgccta atctagaggg cccgtttaaa cccgctgatc agcctcgact
     2221 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2281 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     2341 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2401 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     2461 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     2521 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2581 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2641 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2701 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2761 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2821 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2881 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     2941 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3001 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3061 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     3121 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     3181 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     3241 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     3301 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3361 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3421 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3481 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     3541 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3601 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3661 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3721 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3781 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3841 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3901 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3961 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4021 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     4081 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     4141 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     4201 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4261 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4321 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4381 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     4441 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4501 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4561 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4621 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4681 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4741 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4801 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4861 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4921 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4981 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5041 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5101 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5161 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5221 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5281 gtggcctaac tacggctaca ctagaagaac ggtatttggt atctgcgctc tgctgaagcc
     5341 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5401 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5461 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5521 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5581 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5641 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5701 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5761 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5821 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5881 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5941 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6001 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6061 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6121 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6181 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6241 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6301 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6361 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6421 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6481 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6541 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6601 ccgaaaagtg ccacctg
//
