LOCUS       sequence-430890-        7811 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 7811)
  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 7811)
  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: RXFP1-DuET
FEATURES             Location/Qualifiers
     source          1..7811
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(588..605)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    594..818
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      864..1292
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(951..970)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1161..1182
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1301..1321)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1306..1635
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1486..1621
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1548..1567
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1702..2496
                     /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(1756..1775)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2366..2385
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2670..2791
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2707..2726)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2761..2780
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2840..2856)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2840..2856)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2853..2875)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2864..2880
                     /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(2888..2894,2895..2912,2913..2918))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2933..2954
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3071..3088)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3242..3830)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3322..3341)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4001..4792,4793..4861))
                     /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     4624..4643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4862..4966)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5041..5060)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5232..5611
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5612..5815
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5766..5786
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5860..5879
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5860..5878
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 aatgttttac tcagttcacc agagcgctat taccgcgacc gagatccgga atcaggttaa
       61 gaaggaaatg attctcgcta agaggttctt ttttatagtg tttaccgatg ctctctgttg
      121 gatccctatt ttcgttgtga agtttttgtc tctgctgcag gtggagatcc ctgggactat
      181 cactagctgg gtggtaatct tcatcctccc cataaatagc gccctgaatc ccatcctgta
      241 caccctgacc acgcggccct tcaaggagat gatccaccgc ttttggtaca attatcggca
      301 gcggaagtca atggattcca agggccaaaa gacgtacgca cccagcttta tatgggtgga
      361 aatgtggccc ttgcaggaga tgccgccaga gcttatgaaa cctgacctgt ttacttatcc
      421 atgcgagatg agcctgatct cccagagcac acgcctcaat agttatagta tcgataccgg
      481 tggacgcacc ggtgaattcg atgaggccag caccaccgtg tctaagaccg agacttctca
      541 ggtggcccct gcctaatcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
      601 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
      661 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
      721 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
      781 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
      841 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
      901 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
      961 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1021 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1081 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1141 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1201 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1261 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     1321 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     1381 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     1441 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     1501 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     1561 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     1621 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     1681 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     1741 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     1801 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     1861 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     1921 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     1981 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     2041 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     2101 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     2161 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     2221 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     2281 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     2341 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     2401 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     2461 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     2521 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     2581 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     2641 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     2701 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2761 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     2821 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     2881 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     2941 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3001 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3061 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3121 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3181 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3241 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3301 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3361 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3421 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3481 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3541 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3601 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3661 ctaactacgg ctacactaga agaacggtat ttggtatctg cgctctgctg aagccagtta
     3721 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3781 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3841 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3901 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3961 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4021 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4081 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4141 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4201 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4261 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4321 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4381 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4441 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4501 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4561 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4621 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4681 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4741 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4801 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4861 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4921 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4981 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     5041 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     5101 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacat
     5161 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     5221 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     5281 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     5341 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     5401 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     5461 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     5521 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     5581 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     5641 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     5701 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     5761 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
     5821 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
     5881 gacccaagct ggctagcgtt taaacttaag cttagcggcc gcgccaccat gaagacgatc
     5941 atcgccctga gctacatctt ctgcctggta ttcgccgact acaaggacga tgatgacgcc
     6001 agcatcgata tgacatcagg ctccgtgttc ttctacatcc tgattttcgg taagtatttc
     6061 agccacggcg gcgggcagga cgtgaagtgc agcctcgggt attttccttg tgggaacatc
     6121 acaaagtgcc tcccccagct gctgcactgc aacggagtgg acgattgcgg caatcaggca
     6181 gacgaagaca attgcggtga caataatggg tggagcttgc agtttgataa gtacttcgcc
     6241 agctactata aaatgacctc acaatatcct tttgaggcag agacacccga atgtctggtg
     6301 ggcagcgtcc cggtgcagtg tctgtgtcag gggctcgaac tggactgcga tgagacaaat
     6361 ctgagggctg tgccctctgt tagtagcaac gtcactgcca tgtccttgca atggaacctg
     6421 ataagaaagc tgcctcccga ctgcttcaag aactaccacg acctgcagaa attgtatctc
     6481 caaaataaca aaataacctc tatatcaatc tatgccttta gaggcctcaa ttctttgacg
     6541 aagctctatc tgagccataa tcgaatcaca ttcctcaaac cgggggtgtt tgaggatctg
     6601 catcgcctgg aatggctgat aattgaagat aaccatcttt ctcggattag cccgccaacc
     6661 ttttatgggc ttaatagtct gattttgctc gtgctgatga ataacgtgct tactcgattg
     6721 cctgacaagc ctttgtgtca gcacatgcct cgcttgcatt ggcttgatct ggagggaaac
     6781 catattcaca acttgagaaa cctcaccttc atctcctgca gcaaccttac cgtgctggtc
     6841 atgcgaaaga acaaaattaa ccatctcaac gagaacacct tcgctccact ccagaaactc
     6901 gatgagctgg acctggggtc caataagata gaaaacctcc cccctctgat tttcaaagat
     6961 ctcaaggaac tttcccagct gaatctgtcc tacaatccca ttcagaaaat tcaggccaat
     7021 caattcgact atctggtcaa gttgaagtcc ctgtctctgg aagggattga aatttctaac
     7081 atccagcagc gaatgttcag acccctgatg aacctcagtc acatctattt taagaagttc
     7141 cagtactgtg gatatgcccc tcacgtgagg tcatgcaagc ctaacaccga cggcataagc
     7201 agtctggaga acctcctggc ctccatcatc cagagagtct ttgtctgggt cgtgagtgcc
     7261 gtcacttgct ttggaaacat atttgttata tgtatgagac cctatattag gagcgagaat
     7321 aagctgtacg ccatgagtat tatctcactc tgttgcgccg attgcctgat gggaatatat
     7381 cttttcgtga tcggaggctt cgacctgaaa ttcaggggcg agtataataa gcacgcccaa
     7441 ttgtggatgg aaagtacgca ttgtcagctg gtgggatctc tggctatttt gagcacagag
     7501 gtgtcagtcc tgctgctgac gttccttacc ttggaaaagt acatttgcat tgtgtatcca
     7561 tttcgatgcg tccgcccagg gaagtgtaga acaatcacag tactgatcct catatggatc
     7621 actgggttta ttgtcgcctt catcccgctg agcaataaag agttcttcaa gaattattac
     7681 gggacgaatg gcgtctgttt tcccttgcat tcagaagaca cagaaagcat cggagcacag
     7741 atctattccg tggccatttt ccttgggatc aacctggccg cgttcataat tatcgtgttc
     7801 agctatgggt c
//
