LOCUS       sequence-430899-        8381 bp    DNA     circular SYN 14-APR-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 8381)
  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 8381)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: GABBR1-DuET
FEATURES             Location/Qualifiers
     source          1..8381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1769..1788)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1770..1788)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1833..2036)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1862..1882)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2037..2416
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2588..2607
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2682..2786
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2787..2855,2856..3647)
                     /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(3005..3024)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3818..4406
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4307..4326
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4560..4577
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4694..4715
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4730..4735,4736..4753,4754..4760)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4768..4784
                     /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     4773..4795
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4792..4808
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4792..4808
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4857..4978
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4868..4887)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4922..4941
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5152..5946)
                     /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(5263..5282)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5873..5892
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(6013..6342)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6027..6162
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6081..6100)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     6327..6347
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(6356..6784)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6466..6487)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6678..6697
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6830..7054
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     7043..7060
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 gaggtcgtct tttgtggaat catagtaccc tatcttttta taggaaccac cctgcagctg
       61 ttcgatcaga gtccaggcca tccgtgaccc gcttgcgtcg aagaccacat ggccggagac
      121 gccctcgaaa ctactagagt tcatcgccct gtagatctgg tcggtgattg tttggttgtt
      181 ataattaaag tcctcaagcc tcaccccgga tcggcccccc ccgccggatg tcttattcaa
      241 cgccagggcc aaggcccaga tggcgtcgta ggcgagagga gcctcctgaa atccccctgt
      301 ttcctcaggg tggcgcttca gccgctttgt aagcttctca acaaattctt gagaagtcat
      361 gttggatatg cttctggtgt tagcggggtt caacataaca atttctgttg tgatgtggcc
      421 ctccacggct tccgtcatct cgtctactgt gcagtttatt gaggggtcgt atattttaaa
      481 ccagttatcc gcataccacc caatgaggaa ccacacgtac tttttaccga acaggcgttc
      541 cttgtatacc tcgcaaaaaa ctttccgagc ctccgtttcg tagaacaatc ccacgatgat
      601 tcgggcgtct tgccttttaa ggttcttgac gggcaccgcc gggtcgctaa aaaagctttg
      661 ccggaaagtg atctcaattc ctgcttcctt cactctctcc tcaagatcgt ccagtgtgct
      721 agtaaatacc tcagtagtct gctgaatggt cgcgattttt ttccagcccc atttttcaaa
      781 aagctttacg cgtgtaggat tatgaagggt agcggaaggg tgggtgcgaa agaaggtggg
      841 gaacctctgc cgattagaca aagcggggct ggagctaccg tagctcagaa caatcagatt
      901 ccacattcgt gccgcttctg ccacgagggt actcacagag ctgcagccag gcatcagaat
      961 aattttaatg ggatcgttgt acaagagctc atagagatat ttagttgcct gcccgggatc
     1021 acatttggag tcgtgatgaa tgagtttcag ttcgtaatcg ggcagtatat cccgccggct
     1081 atttacgtct tccagagcca tttcaacagc gggctgacaa gcctgtccac ctggccaccc
     1141 gccggacatg ggaaaaaggg ccccgatgta aacagctcgt ctctcggagt gcggtgtgcg
     1201 attcacttgg cagtggggtt taggggttga ccattggcct tggctgcaaa ttgaccggct
     1261 actgcctacg aggtgaaagt cagggtcgca ccggaaatcc acccgagccc cgtccagagc
     1321 gggaaggtct ccgccggtca gaaagacttt cccattctca agagtcagat aacttttgct
     1381 acaaattctg acacacctgc ttggtgtatc catatccgtc caacttccat tggcaagaca
     1441 tttgcgcacc ttaggtccaa ccacctccct ttcgcctctg cagacgtatt cgatctcata
     1501 atccaccgga aggaaattga tggccttgac ctgatcccgt gtcaagccgc ggtatcttat
     1561 ccccccctcc caaggcggat gaataatttg gcagccctca cttgtagcgt taggtgtctg
     1621 cgccccccct gcccctggat cgatgctggc gtcatcatcg tccttgtagt cggcgaatac
     1681 caggcagaag atgtagctca gggcgatgat cgtcttcatg gtggcgcggc cgctaagctt
     1741 aagtttaaac gctagccagc ttgggtctcc ctatagtgag tcgtattaat ttcgataagc
     1801 cagtaagcag tgggttctct agttagccag agagctctgc ttatatagac ctcccaccgt
     1861 acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt tggaaagtcc
     1921 cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag acttggaaat
     1981 ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg catcaccatg
     2041 gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca taaggtcatg
     2101 tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt
     2161 ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat
     2221 tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat tattgacgtc
     2281 aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg
     2341 cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta
     2401 gtcaataatc aatgtcaacg cgtatatctg gcccgtacat cgcgaagcag cgcaaaacgc
     2461 ctaaccctaa gcagattctt catgcaaatg tcggtcaagc cttgccttgt tgtagcttaa
     2521 attttgctcg cgcactactc agcgacctcc aacacacaag cagggagcag atactggctt
     2581 aactatgcgg catcagagca gattgtactg agagtgcacc ataggggatc gggagatctc
     2641 ccgatccgtc gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2701 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2761 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     2821 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     2881 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2941 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3001 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     3061 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3121 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3181 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3241 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3301 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3361 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3421 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3481 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3541 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3601 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     3661 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     3721 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3781 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3841 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3901 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3961 ccgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4021 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4081 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4141 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4201 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4261 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4321 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4381 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4441 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4501 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4561 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     4621 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     4681 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     4741 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     4801 gctatgacca tgattacgcc aagctctagc tagaggtcga cggtatacag acatgataag
     4861 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     4921 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg
     4981 ggtgggcgaa gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc
     5041 ggaaaacgat tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgtg
     5101 atggcaggtt gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg ctcagaagaa
     5161 ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag
     5221 cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa
     5281 cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa
     5341 gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc
     5401 ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg
     5461 atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg
     5521 ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag
     5581 ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag
     5641 gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac
     5701 gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc
     5761 gtcctgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc
     5821 ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc
     5881 atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc
     5941 aatcatgcga aacgatcctc atcctgtctc ttgatcagat ccgaaaatgg atatacaagc
     6001 tcccgggagc tttttgcaaa agcctaggcc tccaaaaaag cctcctcact acttctggaa
     6061 tagctcagag gcagaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg
     6121 gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg
     6181 gactatggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     6241 tggggacttt ccacacctgg ttgctgacta attgagatgc atgctttgca tacttctgcc
     6301 tgctggggag cctggggact ttccacaccc taactgacac acattccaca gaattaattc
     6361 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     6421 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     6481 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     6541 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     6601 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     6661 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     6721 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     6781 gcgtggggat accccctaga gccccagctg gttctttccg cctcagaagc catagagccc
     6841 accgcatccc cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc
     6901 accccacccc ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt
     6961 tattaggaaa ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg
     7021 caaacaacag atggctggca actagaaggc acagtcgagg ctgatcagcg ggtttaaacg
     7081 ggccctctag attaggcagg ggccacctga gaagtctcgg tcttagacac ggtggtgctg
     7141 gcctcatcga attcaccggt gcgtccaccg gtatcgattt tatacaggag gtgaacgcga
     7201 cttccatcgc agctcagcct gtcaggtggc tctggggggc ctcgtggaag tcccccgcta
     7261 ggctctggtg gggtaggagg atggcggcgg ctgcgcaatt gctgcctgct ttgcagctga
     7321 tgcctcagct cagagacgcg ctcctccttc tctgcaatga ttttctccag ttccctattc
     7381 tccttttcga ggagcctaga cttttcctct tcgttattgt tagtagatga gcctgttttc
     7441 atggtatcct gggcctcact ttgccattct cccctcgtta tcagtcgtct catctttggt
     7501 acaaaaagta ccaccagtgt aatgtagctg ctaaacacga tggccagaga tgcgaaagcg
     7561 aaggctgcat cctgctgaga ggacaaaatc attgtcactg gtgcggtgat gagacacagg
     7621 acggccacat tgtagatagc catccccacc gccctgtgat cgttgatctt ttcggtggac
     7681 acgctcttgg tttcgtatgc caggaaaatc ccgagcagca aaagcaggcc cttgtacccg
     7741 tagaaaatgc ccaaccatgt gttcatcttc ctgctactgc agtgctccaa ctggggcaga
     7801 attgatacgt cgatatcctc cttcggctcc tctttggcga aagtttcaat ggttctgtgc
     7861 aggggatcaa ctatctgcca gattgcgaga gtcaggacgt ccattcccac caacaggccc
     7921 acggttgcat agagtttcca cggttcgagc gtcttgcgcc attccttctt ctcctctttt
     7981 tttgtaaaga cagtatgaac ccaccagatc ttagtgaaca tggatccata acccagactg
     8041 aatcccagtc ccagcaacca cagccgagcc tggcaaacga aaggaaactg atttcggccg
     8101 atgtgatagc cgtccaagcc caaaggaaac acggcggcca atgcgagaga gcatcccaca
     8161 gcggtaaggt tattgaggtt aggctgggag ttctggatat atctgacgtg agagttgtag
     8221 atgttaaagg acagacagac aacggcgagc acgatgccca gagaagacag gacagacaca
     8281 gaaataaaca gcttctgtga gaggaacctg aaagtcttaa tcacgagggt ctggtcagca
     8341 ggtgggctcc ccccaatcca cttatcggtc ttggaccagg a
//
