LOCUS       sequence-430894-        6920 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 6920)
  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 6920)
  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: CHRM1-DuET
FEATURES             Location/Qualifiers
     source          1..6920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     462..481
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(700..804)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(879..898)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1070..1449
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1450..1653
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1604..1624
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1698..1717
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1698..1716
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3346..3363)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3352..3576
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3622..4050
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3709..3728)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3919..3940
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4059..4079)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4064..4393
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4244..4379
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4306..4325
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4460..5254
                     /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(4514..4533)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5124..5143
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5428..5549
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5465..5484)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5519..5538
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5598..5614)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5598..5614)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5611..5633)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5622..5638
                     /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(5646..5652,5653..5670,5671..5676))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5691..5712
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5829..5846)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6000..6588)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6080..6099)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6759..6920,1..630,631..699))
                     /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 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
       61 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
      121 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
      181 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
      241 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
      301 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
      361 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
      421 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
      481 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
      541 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
      601 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
      661 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
      721 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
      781 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
      841 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
      901 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
      961 aaaatttaag ctacaacaag gcaaggcttg accgacattt gcatgaagaa tctgcttagg
     1021 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
     1081 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     1141 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     1201 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     1261 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     1321 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     1381 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     1441 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
     1501 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     1561 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
     1621 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
     1681 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagcgttta
     1741 aacttaagct tagcggccgc gccaccatga agacgatcat cgccctgagc tacatcttct
     1801 gcctggtatt cgccgactac aaggacgatg atgacgccag catcgatatg aatacaagcg
     1861 ctcctccagc ggtttcccca aatattacgg tgctggcacc tgggaaagga ccatggcaag
     1921 ttgcttttat cggtatcacc accggcttgc tctccttggc aacagtcacg ggcaatttgc
     1981 tcgtactgat ctcatttaag gtgaacacag agctgaaaac agtgaacaac tacttcttgt
     2041 tgagtttggc ctgtgcggac cttattatcg gaacgttttc aatgaatctg tatacaacct
     2101 atctcctgat gggccactgg gcgctgggaa cccttgcttg cgacctttgg ttggcccttg
     2161 attacgtggc gagcaacgca agcgtcatga atctcctgtt gatatcattt gacaggtatt
     2221 tttctgttac ccggcccctg agctacagag ctaaacgaac tccacgacgc gctgcactga
     2281 tgattggtct ggcctggctg gtcagttttg tcctgtgggc ccctgccata ctgttttggc
     2341 agtacctggt gggagagcgc accgtgcttg caggtcagtg ctatatacag tttctgtctc
     2401 agcccataat cacattcggc accgcgatgg cggcatttta cttgcctgtg actgttatgt
     2461 gtactctgta ttggcgaatt taccgcgaaa ccgagaatag agccagggaa cttgctgcac
     2521 tgcagggttc agagacaccc ggaaagggcg gcggctccag tagtagcagt gagagaagtc
     2581 aacctggggc tgaagggtcc cccgagactc ccccaggaag gtgctgccga tgctgcaggg
     2641 cccctagatt gttgcaggcc tactcttgga aggaggaaga ggaggaagac gaaggctcca
     2701 tggagagtct cacctcttca gagggggaag agccagggag cgaggtggtt atcaagatgc
     2761 caatggtgga cccagaagcg caggcaccaa caaaacagcc cccacgaagt agccccaaca
     2821 ccgttaaaag accaactaaa aaggggaggg atcgcgcagg caagggtcaa aaacccaggg
     2881 gaaaggaaca gctggcgaag cggaaaacct tcagcctcgt gaaggaaaag aaagccgcac
     2941 gaaccctgtc cgctatcttg ctggctttca tactgacctg gacgccttat aacattatgg
     3001 tgctggtctc caccttttgt aaagattgcg tcccagagac actttgggag ctggggtact
     3061 ggctgtgcta tgtgaactcc acaattaacc caatgtgtta tgccttgtgt aataaagcgt
     3121 tcagagatac attccggctt ctccttctgt gccggtggga taagaggcgc tggcgcaaaa
     3181 tccccaaacg gcccgggtca gttcacagaa cacctagcag acaatgtatc gataccggtg
     3241 gacgcaccgg tgaattcgat gaggccagca ccaccgtgtc taagaccgag acttctcagg
     3301 tggcccctgc ctaatctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     3361 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3421 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3481 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3541 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3601 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3661 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3721 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3781 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3841 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3901 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3961 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4021 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     4081 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4141 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4201 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     4261 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4321 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4381 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     4441 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     4501 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     4561 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     4621 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     4681 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     4741 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4801 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4861 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4921 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4981 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     5041 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     5101 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     5161 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     5221 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     5281 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     5341 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     5401 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     5461 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     5521 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     5581 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     5641 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     5701 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5761 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5821 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5881 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5941 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6001 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6061 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6121 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6181 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6241 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6301 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6361 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6421 aactacggct acactagaag aacggtattt ggtatctgcg ctctgctgaa gccagttacc
     6481 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6541 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6601 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6661 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6721 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6781 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6841 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6901 gacccacgct caccggctcc
//
