LOCUS       sequence-430764-        6596 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 6596)
  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 6596)
  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: CCR5-DuET
FEATURES             Location/Qualifiers
     source          1..6596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      101..689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     590..609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     843..860
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    977..998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1013..1018,1019..1036,1037..1043)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1051..1067
                     /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     1056..1078
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1075..1091
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1075..1091
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1140..1261
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1151..1170)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1205..1224
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1435..2229)
                     /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(1546..1565)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2156..2175
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2296..2625)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2310..2445
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2364..2383)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2610..2630
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2639..3067)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2749..2770)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2961..2980
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3113..3337
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3326..3343
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4648..4667)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4649..4667)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4712..4915)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4741..4761)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4916..5295
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5467..5486
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5561..5665
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5666..5734,5735..6526)
                     /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(5884..5903)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
       61 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
      121 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
      181 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
      241 ataccgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
      301 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
      361 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
      421 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
      481 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
      541 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
      601 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
      661 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
      721 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
      781 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
      841 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
      901 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
      961 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     1021 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     1081 acagctatga ccatgattac gccaagctct agctagaggt cgacggtata cagacatgat
     1141 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     1201 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     1261 tggggtgggc gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt
     1321 cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc
     1381 gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa ccccagagtc ccgctcagaa
     1441 gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta
     1501 aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc
     1561 caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga
     1621 aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag
     1681 atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc
     1741 ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc
     1801 tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg
     1861 cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga
     1921 caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac
     1981 aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc
     2041 ctcgtcctgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg
     2101 cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca
     2161 gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg
     2221 ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gatccgaaaa tggatataca
     2281 agctcccggg agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg
     2341 gaatagctca gaggcagagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc
     2401 atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg
     2461 cgggactatg gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga
     2521 gcctggggac tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct
     2581 gcctgctggg gagcctgggg actttccaca ccctaactga cacacattcc acagaattaa
     2641 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     2701 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     2761 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     2821 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     2881 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     2941 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     3001 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3061 ggcgcgtggg gataccccct agagccccag ctggttcttt ccgcctcaga agccatagag
     3121 cccaccgcat ccccagcatg cctgctattg tcttcccaat cctccccctt gctgtcctgc
     3181 cccaccccac cccccagaat agaatgacac ctactcagac aatgcgatgc aatttcctca
     3241 ttttattagg aaaggacagt gggagtggca ccttccaggg tcaaggaagg cacgggggag
     3301 gggcaaacaa cagatggctg gcaactagaa ggcacagtcg aggctgatca gcgggtttaa
     3361 acgggccctc tagactcgag ttaggcaggg gccacctgag aagtctcggt cttagacacg
     3421 gtggtgctgg cctcatcgaa ttcaccggtg cgtccaccgg tatcgatcag tccgacagaa
     3481 atctcttgct cccccgtgct gcgcgtataa actgaactag cccgctcggg tgcctcctgt
     3541 tgaaagatac tgcaacactt acaaaatctt ttagcaatgt gcttctggaa aaagaccaga
     3601 aggtagtttc taaacttctc gccgacgaaa gcgtagatga taggatttat gcaacaatgc
     3661 gtcatgccaa gtgtctcggt gacctgcatg gcctgatcca gcctgttgga gctagagcag
     3721 ttattcagac caaaaaattc ctgaaaggta ttcagcaaaa gtacaatgtt ataaggcgcc
     3781 caaaaaagaa agtagacaat cattatagtg aagatcaggc ggactgcccg atgccgcttc
     3841 ttctcgttgc ggcaccgcag gagcgtcttc aaaatgcctg aatagcatat caccatcacg
     3901 agcagaggca acacgagtcc gaggatcacg attttcaggg tctggaaatt tttccagaac
     3961 tgatactgag aataagggaa gtgtgagctg catgtatagt gcaggccttc cttctggctg
     4021 cgggtaaaga tgatgccggg caggctggca aatactgcaa ccacccaggt tatcactgaa
     4081 gttaccaccc caaaggtgac cgtccgggcc ttcagggcga agactgcgtg gaccacggcc
     4141 aggtatctat ctatagtgag gaggatgatg aagaatattc cgctaaagaa tccaatgaaa
     4201 tacaatccgg taagcagttg gcacattgtg tttccaaagt cccactgagc ggctgcgtag
     4261 tgcgcccaga agggcacagt cagcagaaag aacaaatcgc ttatagccag gttcaggagg
     4321 tagatatctg tcatgctttt caacctttta cagttgatga gaatcagaat caccaacata
     4381 ttacctacaa atccgaaaat gaataccaat gagtacaggg gaggaagcag ccgggctgca
     4441 atctgcttga catttatctt ctggcatggt tcgcttgtat aatagttaat atcatagatg
     4501 ggagaactca cctgataatc catatcgatg ctggcgtcat catcgtcctt gtagtcggcg
     4561 aataccaggc agaagatgta gctcagggcg atgatcgtct tcatggtggc gctaagctta
     4621 agtttaaacg ctagccagct tgggtctccc tatagtgagt cgtattaatt tcgataagcc
     4681 agtaagcagt gggttctcta gttagccaga gagctctgct tatatagacc tcccaccgta
     4741 cacgcctacc gcccatttgc gtcaatgggg cggagttgtt acgacatttt ggaaagtccc
     4801 gttgattttg gtgccaaaac aaactcccat tgacgtcaat ggggtggaga cttggaaatc
     4861 cccgtgagtc aaaccgctat ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg
     4921 taatagcgat gactaatacg tagatgtact gccaagtagg aaagtcccat aaggtcatgt
     4981 actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg ggcgtacttg
     5041 gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac tccacccatt
     5101 gacgtcaatg gaaagtccct attggcgtta ctatgggaac atacgtcatt attgacgtca
     5161 atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt tatgtaacgc
     5221 ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat taataactag
     5281 tcaataatca atgtcaacgc gtatatctgg cccgtacatc gcgaagcagc gcaaaacgcc
     5341 taaccctaag cagattcttc atgcaaatgt cggtcaagcc ttgccttgtt gtagcttaaa
     5401 ttttgctcgc gcactactca gcgacctcca acacacaagc agggagcaga tactggctta
     5461 actatgcggc atcagagcag attgtactga gagtgcacca taggggatcg ggagatctcc
     5521 cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5581 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5641 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5701 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5761 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5821 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5881 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5941 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     6001 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     6061 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6121 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6181 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6241 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6301 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6361 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6421 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6481 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6541 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatct
//
