LOCUS       sequence-430765-        6662 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 6662)
  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 6662)
  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: CCR6-DuET
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     419..438
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(657..761)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(836..855)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1027..1406
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1407..1610
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1561..1581
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1655..1674
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1655..1673
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3045..3062)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3051..3275
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3321..3749
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3408..3427)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3618..3639
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3758..3778)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3763..4092
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3943..4078
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4005..4024
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4159..4953
                     /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(4213..4232)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4823..4842
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5127..5248
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5164..5183)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5218..5237
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5297..5313)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5297..5313)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5310..5332)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5321..5337
                     /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(5345..5351,5352..5369,5370..5375))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5390..5411
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5528..5545)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5699..6287)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5779..5798)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6458..6662,1..587,588..656))
                     /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 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
       61 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
      121 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
      181 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
      241 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
      301 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
      361 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
      421 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
      481 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
      541 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
      601 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
      661 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
      721 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
      781 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
      841 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      901 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacatttgca
      961 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
     1021 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     1081 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     1141 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     1201 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     1261 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     1321 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     1381 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     1441 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     1501 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     1561 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
     1621 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
     1681 aagctggcta gcgtttaaac ttaagcttag cgccaccatg aagacgatca tcgccctgag
     1741 ctacatcttc tgcctggtat tcgccgacta caaggacgat gatgacgcca gcatcgatat
     1801 gagcggagag agcatgaact tcagtgatgt gttcgattcc agtgaggact acttcgtgtc
     1861 cgtcaataca tcctactaca gcgtagattc tgagatgctg ctttgctccc tgcaggaggt
     1921 gcgccagttc tcccgactct tcgttcccat cgcgtattca ctcatctgcg tctttggcct
     1981 cctggggaat atcttggtag tcatcacatt tgctttttat aaaaaggcca gatccatgac
     2041 agacgtctac ctgcttaata tggccattgc agacatcctg ttcgtcctga cgctcccatt
     2101 ctgggctgtg agtcacgcta ccggggcctg ggtgttcagt aatgcaacct gcaagctgtt
     2161 gaagggaatt tatgccatca actttaattg cggtatgctg ctgctgactt gcataagtat
     2221 ggacagatac atcgccatag tacaggcaac aaaatccttc agactgaggt ctcgaacgct
     2281 ccctaggtct aaaatcattt gtcttgttgt atggggcttg agcgtgatta tttccagcag
     2341 tactttcgtg tttaaccaga agtacaacac gcagggcagc gatgtgtgtg agcccaagta
     2401 ccagacggta tctgagccta tcaggtggaa gctgttgatg ctgggtttgg aattgctgtt
     2461 cggctttttt atcccactca tgtttatgat tttttgttat accttcatcg tgaagaccct
     2521 ggtccaggca caaaactcta agcgacacaa ggccatccgg gtaatcatcg ctgttgtgct
     2581 ggtatttctg gcttgccaga tccctcataa tatggtcctt ctggtaacgg cagccaacct
     2641 gggcaaaatg aatcgctcat gtcagtcaga aaagcttatc gggtacacca aaacggttac
     2701 ggaggttctg gcgttcctgc attgttgcct taaccctgtg ctgtacgcat ttataggtca
     2761 gaagtttcga aactacttcc tgaagattct caaagacctc tggtgtgtaa gaagaaagta
     2821 taaaagtagt ggattttcat gcgcaggacg ctactccgaa aacatttcca gacagaccag
     2881 tgagacggcc gataacgata acgcctcctc ctttaccatg atcgataccg gtggacgcac
     2941 cggtgaattc gatgaggcca gcaccaccgt gtctaagacc gagacttctc aggtggcccc
     3001 tgcctaactc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     3061 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3121 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3181 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3241 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3301 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3361 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3421 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3481 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3541 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3601 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3661 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3721 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3781 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3841 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3901 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3961 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4021 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4081 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     4141 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     4201 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     4261 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     4321 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     4381 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4441 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     4501 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     4561 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4621 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4681 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4741 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4801 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4861 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4921 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4981 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     5041 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     5101 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     5161 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5221 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     5281 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5341 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5401 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5461 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5521 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5581 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5641 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5701 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5761 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5821 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5881 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5941 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6001 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6061 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6121 actacggcta cactagaaga acggtatttg gtatctgcgc tctgctgaag ccagttacct
     6181 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6241 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6301 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6361 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6421 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6481 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6541 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6601 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6661 gc
//
