LOCUS       sequence-430777-        6872 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 6872)
  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 6872)
  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: CRHR1-DuET
FEATURES             Location/Qualifiers
     source          1..6872
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     557..576
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(795..899)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(974..993)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1165..1544
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1545..1748
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1699..1719
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1793..1812
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1793..1811
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3393..3410)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3399..3623
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3669..4097
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3756..3775)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3966..3987
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4106..4126)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4111..4440
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4291..4426
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4353..4372
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4507..5301
                     /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(4561..4580)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5171..5190
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5475..5596
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5512..5531)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5566..5585
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5645..5661)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5645..5661)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5658..5680)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5669..5685
                     /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(5693..5699,5700..5717,5718..5723))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5738..5759
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5876..5893)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6047..6635)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6127..6146)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6806..6872,1..725,726..794))
                     /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 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
       61 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
      121 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
      181 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
      241 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
      301 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
      361 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
      421 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
      481 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
      541 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
      601 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
      661 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
      721 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
      781 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
      841 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
      901 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
      961 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     1021 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
     1081 gcttgaccga catttgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
     1141 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
     1201 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     1261 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1321 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     1381 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     1441 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     1501 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     1561 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     1621 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     1681 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     1741 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
     1801 ctcactatag ggagacccaa gctggctagc gtttaaactt aagcttagcg ccaccatgaa
     1861 gacgatcatc gccctgagct acatcttctg cctggtattc gccgactaca aggacgatga
     1921 tgacgccagc atcgatatgg gaggtcatcc gcagctgcgg cttgttaagg ctctcctgct
     1981 gctgggcctc aaccctgtca gtgccagcct ccaggaccag cactgtgagt ctctcagtct
     2041 ggccagcaat ataagtggac tgcagtgtaa cgcctcagtg gacttgatag gcacttgctg
     2101 gcctcgaagt cctgctggtc agctggtggt acgaccctgc ccagcattct tctatggcgt
     2161 gcgatataac accacgaata atgggtaccg cgaatgcctg gccaacggca gttgggccgc
     2221 aagggtgaac tattctgaat gccaggaaat tctgaatgaa gaaaagaaga gtaaggttca
     2281 ctaccacgtg gccgtgatca tcaactatct cggtcactgt atctcactgg ttgccttgtt
     2341 ggttgcattc gtactgtttt tgaggctgag acctggctgt acacactggg gagaccaggc
     2401 tgatggagcc ctggaggtgg gggccccttg gtccggagca ccttttcaag tccgacgcag
     2461 catccggtgc ctgcgcaaca taatccactg gaaccttata tccgctttca ttctgaggaa
     2521 cgctacatgg ttcgtggtgc agttgactat gtctcctgag gtccatcagt ccaacgttgg
     2581 ctggtgtagg ctggtcacag ctgcatacaa ttacttccat gtgaccaatt tcttttggat
     2641 gttcggtgaa gggtgctatc tccacacggc aatagttctg acgtactcaa cagaccgact
     2701 tagaaaatgg atgttcatct gcattgggtg gggtgtccca tttcccatca tcgttgcctg
     2761 ggctataggc aagctctact acgataatga aaagtgttgg tttggtaagc ggccaggcgt
     2821 atacaccgac tacatctatc agggccccat gatactggtg ctgctgatca acttcatttt
     2881 tttgtttaac atcgtgagga tcctcatgac gaaattgcga gcatccacta cttctgagac
     2941 aatccaatac cggaaagccg tgaaggctac cctggtactg ctgcctctgc tgggcatcac
     3001 ttacatgctg tttttcgtga accctggcga ggatgaagtc agtagggtgg tgtttattta
     3061 tttcaactcc ttcttggagt ccttccaggg gttctttgtg tctgtctttt actgcttttt
     3121 gaatagcgaa gtcagaagtg ctattcggaa gcgatggcac agatggcagg acaaacattc
     3181 tatcagagct agggtggcac gggcaatgtc catccccacc tccccaacca gggtttcctt
     3241 tcactcaatc aagcagagta ccgccgtgat cgataccggt ggacgcaccg gtgaattcga
     3301 tgaggccagc accaccgtgt ctaagaccga gacttctcag gtggcccctg cctaactcga
     3361 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     3421 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3481 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3541 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3601 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3661 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3721 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3781 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3841 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3901 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3961 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     4021 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     4081 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     4141 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     4201 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4261 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4321 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     4381 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4441 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     4501 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4561 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4621 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4681 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4741 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4801 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4861 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4921 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4981 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     5041 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     5101 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     5161 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     5221 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     5281 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     5341 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5401 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5461 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5521 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5581 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     5641 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5701 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5761 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5821 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5881 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5941 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     6001 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     6061 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     6121 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6181 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6241 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6301 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6361 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6421 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6481 ctagaagaac ggtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6541 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6601 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6661 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6721 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6781 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6841 cgatctgtct atttcgttca tccatagttg cc
//
