LOCUS       sequence-430871-        6407 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 6407)
  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 6407)
  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: MRGPRG-DuET
FEATURES             Location/Qualifiers
     source          1..6407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     552..571
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(790..894)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(969..988)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1160..1539
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1540..1743
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1694..1714
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1788..1807
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1788..1806
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2923..2940)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2929..3153
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3199..3627
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3286..3305)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3496..3517
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3636..3656)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3641..3970
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3821..3956
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3883..3902
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4037..4831
                     /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(4091..4110)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4701..4720
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5005..5126
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5042..5061)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5096..5115
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5175..5191)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5175..5191)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5188..5210)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5199..5215
                     /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(5223..5229,5230..5247,5248..5253))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5268..5289
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5406..5423)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5577..6165)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5657..5676)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6336..6407,1..720,721..789))
                     /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"
ORIGIN
        1 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
       61 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
      121 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
      181 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
      241 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
      301 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
      361 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
      421 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
      481 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
      541 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
      601 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
      661 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
      721 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
      781 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
      841 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
      901 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
      961 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
     1021 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     1081 accgacattt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     1141 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
     1201 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     1261 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     1321 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     1381 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     1441 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     1501 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     1561 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     1621 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     1681 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     1741 gctctctggc taactagaga acccactgct tactggctta tcgaaattaa tacgactcac
     1801 tatagggaga cccaagctgg ctagcgttta aacttaagct tagcgccacc atgaagacga
     1861 tcatcgccct gagctacatc ttctgcctgg tattcgccga ctacaaggac gatgatgacg
     1921 ccagcatcga tatgttcggc cttttcggcc tgtggcgcac gttcgatagc gtagtgtttt
     1981 atctcacttt gattgttggc ctcggcggcc ctgtgggcaa cggcctggta ctgtggaacc
     2041 tcggtttcag aataaaaaaa ggtccattca gcatttacct tctgcatctg gccgccgctg
     2101 atttcctttt cctcagttgc cgggtaggct ttagcgtggc tcaggcagcc cttggagcgc
     2161 aggatacact gtacttcgtg ctcacgttct tgtggtttgc tgtgggcctt tggctgctgg
     2221 ccgccttttc agttgaacgg tgtctctccg atctgttccc agcctgctac cagggttgtc
     2281 ggcctcgcca cgccagcgcc gtgctctgtg cgctggtctg gaccccaact ctgcccgcag
     2341 ttccactgcc cgctaatgcc tgcggtctgc ttcgcaactc agcatgccca ctggtttgtc
     2401 ctcgctacca cgtggcctct gtgacatggt ttctggtgtt ggcccgggtt gcctggaccg
     2461 ccggcgtggt tctgtttgtg tgggtcacct gttgtagcac gcgaccccgc ccccggctgt
     2521 acggaatagt tcttggtgcc cttctgttgt tgtttttttg tgggctgccc tctgtgttct
     2581 actggagcct tcagcctctg ttgaacttcc tgctgcctgt tttctcccca ctcgccaccc
     2641 tgctcgcatg tgtgaactct agcagcaaac ccttgatata ttccgggctg ggccggcaac
     2701 ctggtaagcg cgagccgctt cggtctgtct tgcgcagggc actgggtgag ggagcagaac
     2761 tcggcgctag agggcagtct cttcccatgg gattgctgat cgataccggt ggacgcaccg
     2821 gtgaattcga tgaggccagc accaccgtgt ctaagaccga gacttctcag gtggcccctg
     2881 cctaactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     2941 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3001 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3061 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3121 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     3181 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3241 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3301 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3361 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3421 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3481 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3541 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3601 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     3661 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3721 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3781 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3841 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3901 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3961 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     4021 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4081 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4141 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4201 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4261 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4321 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4381 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4441 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4501 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4561 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     4621 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4681 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4741 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4801 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4861 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4921 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4981 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     5041 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5101 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     5161 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5221 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     5281 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5341 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5401 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5461 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5521 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5581 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5641 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5701 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5761 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5821 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5881 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5941 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6001 tacggctaca ctagaagaac ggtatttggt atctgcgctc tgctgaagcc agttaccttc
     6061 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6121 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6181 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6241 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6301 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6361 cctatctcag cgatctgtct atttcgttca tccatagttg cctgact
//
