LOCUS       sequence-431751-        6857 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 6857)
  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 6857)
  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: CALCRL-DuET
FEATURES             Location/Qualifiers
     source          1..6857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      214..802
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     703..722
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     956..973
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1090..1111
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1126..1131,1132..1149,1150..1156)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1164..1180
                     /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     1169..1191
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1188..1204
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1188..1204
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1253..1374
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1264..1283)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1318..1337
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1548..2342)
                     /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(1659..1678)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2269..2288
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2409..2738)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2423..2558
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2477..2496)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2723..2743
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2752..3180)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2862..2883)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3074..3093
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3226..3450
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3439..3456
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5022..5041)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5023..5041)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5086..5289)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5115..5135)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5290..5669
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5841..5860
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5935..6039
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6040..6108,6109..6857,1..43)
                     /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(6258..6277)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
       61 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
      121 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
      181 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
      241 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
      301 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaataccgt
      361 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
      421 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
      481 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
      541 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
      601 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
      661 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
      721 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      781 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      841 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      901 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      961 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     1021 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     1081 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     1141 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     1201 tgaccatgat tacgccaagc tctagctaga ggtcgacggt atacagacat gataagatac
     1261 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     1321 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg
     1381 ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa
     1441 aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg
     1501 caggttgggc gtcgcttggt cggtcatttc gaaccccaga gtcccgctca gaagaactcg
     1561 tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg
     1621 aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct
     1681 atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg
     1741 ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg
     1801 ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc
     1861 tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg
     1921 atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc
     1981 cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga
     2041 tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg
     2101 agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc
     2161 tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc
     2221 gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag
     2281 ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc
     2341 atgcgaaacg atcctcatcc tgtctcttga tcagatccga aaatggatat acaagctccc
     2401 gggagctttt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc
     2461 tcagaggcag aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc
     2521 ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact
     2581 atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     2641 gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct
     2701 ggggagcctg gggactttcc acaccctaac tgacacacat tccacagaat taattcgcgt
     2761 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     2821 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     2881 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     2941 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3001 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3061 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3121 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3181 ggggataccc cctagagccc cagctggttc tttccgcctc agaagccata gagcccaccg
     3241 catccccagc atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc
     3301 caccccccag aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt
     3361 aggaaaggac agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa
     3421 caacagatgg ctggcaacta gaaggcacag tcgaggctga tcagcgggtt taaacgggcc
     3481 ctctagactc gagttaggca ggggccacct gagaagtctc ggtcttagac acggtggtgc
     3541 tggcctcatc gaattcaccg gtgcgtccac cggtatcgat gttgtacaga ttttctggct
     3601 tcagcaggac gttctcaata tcgtgaatag atttcccgtt aagatgttca gatgggcagt
     3661 catgagagta gcccggccca tcgcttatgg tacttactgt gtaactggcg gacctgaggg
     3721 cttcactatt tgaaaagcta ttgccaaact gtatcttgta ttgattccaa ttccgccgga
     3781 gtatggcctg cacttcaccg ttaaaaaagc agaaaatggt gctcaccaga agcccctgga
     3841 aatgcatgag aatgtgcata atgtaatcgt acacttcctc tgcgattttt ccttcaggcc
     3901 tccatggaat cagcacgaat tcgataccca gcagaggtac caagatcaaa gtggctctga
     3961 cggccttcat gtacaggttg ctttctgcct gatgagtgac cttcaatttt gtgatcagca
     4021 ctctcactat gttcagaagg aagaacagat tgaccagcag agcggcgcag attgggccat
     4081 gaataatgta gagcaggtgc gtgtcagatg agatccaaca gttatcgttg tagtacagtg
     4141 acctagcgat cgcatgaata caggccggaa tcaaagggaa cccccacccc aaaaagtagt
     4201 accacataag atgctgcttc tcagcgaaca cagccaccac tatcaatgta tgaagatata
     4261 taccctcaca gagcatccag aagtagttgc agcccatcag gtacaagtga atgaattggg
     4321 agactttgca agacacggga ttagtggcga ccagcgcctg gttattggcg accgctgtca
     4381 aatgtatgat agtcactacg ctattgcaaa cgaatgagaa gaacaggttt ttatggagtg
     4441 taatcctctg gcagctcaag cttttaaaat agaagaatat gcccagagaa atgaggaggg
     4501 aggctattga gagaccgtgg cctatgattg tgaggtaaaa caggttcagg gcagttttga
     4561 ctttttcgtg ggtattcaca ttgcactgtg tgtaatttgt ccatgtgcgg tttgatgcgg
     4621 ggtgcctaaa ccaattgcca tcctgatcac aaatcttagt tactttttcg gatgggtcaa
     4681 aatcctggaa ataatctgga cagagctgca tagactctgt gccggcggcg acgtcattcc
     4741 agcacaacca tccgtcccac gtgcggttgc aatagacccc ttctgcttgc tggatagggt
     4801 cctgcattat cttctggtaa cactcgtatt gtgctgtcat gattttgttc cgcgtgactc
     4861 ccagctggat tgagtcctcg ggactctctt ccagctcatc gatgctggcg tcatcatcgt
     4921 ccttgtagtc ggcgaatacc aggcagaaga tgtagctcag ggcgatgatc gtcttcatgg
     4981 tggcgctaag cttaagttta aacgctagcc agcttgggtc tccctatagt gagtcgtatt
     5041 aatttcgata agccagtaag cagtgggttc tctagttagc cagagagctc tgcttatata
     5101 gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca
     5161 ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg
     5221 gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa
     5281 ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc
     5341 ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat
     5401 agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa
     5461 atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt
     5521 cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt
     5581 aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta
     5641 ctattaataa ctagtcaata atcaatgtca acgcgtatat ctggcccgta catcgcgaag
     5701 cagcgcaaaa cgcctaaccc taagcagatt cttcatgcaa atgtcggtca agccttgcct
     5761 tgttgtagct taaattttgc tcgcgcacta ctcagcgacc tccaacacac aagcagggag
     5821 cagatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatagggg
     5881 atcgggagat ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5941 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6001 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6061 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6121 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6181 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6241 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6301 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6361 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6421 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6481 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6541 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6601 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6661 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6721 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6781 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6841 atggatgaac gaaatag
//
