LOCUS       sequence-430816-        6497 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 6497)
  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 6497)
  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: GPR171-DuET
FEATURES             Location/Qualifiers
     source          1..6497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        114..317
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     268..288
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     362..381
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        362..380
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1035..1043
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(1587..1604)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1593..1817
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1863..2291
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1950..1969)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2160..2181
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2300..2320)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2305..2634
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2485..2620
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2547..2566
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2701..3495
                     /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(2755..2774)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3365..3384
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3669..3790
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3706..3725)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3760..3779
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3839..3855)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3839..3855)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3852..3874)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3863..3879
                     /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(3887..3893,3894..3911,3912..3917))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3932..3953
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4070..4087)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4241..4829)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4321..4340)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5000..5791,5792..5860))
                     /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     5623..5642
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5861..5965)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6040..6059)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6231..6497,1..113)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
       61 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      121 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      181 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      241 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      301 tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa
      361 ttaatacgac tcactatagg gagacccaag ctggctagcg tttaaactta agcttagcgc
      421 caccatgaag acgatcatcg ccctgagcta catcttctgc ctggtattcg ccgactacaa
      481 ggacgatgat gacgccagca tcgatatgac aaactcttct tttttctgtc ctgtctacaa
      541 ggatctggag cccttcacct acttttttta cctcgtcttt ctggtgggta tcatcggtag
      601 ctgctttgcc acttgggctt tcatccagaa gaacacgaat caccgctgcg tgtcaatcta
      661 tttgataaac cttctgaccg cggactttct tctgacgctg gcccttcccg tcaaaattgt
      721 cgtagacttg ggcgttgccc cgtggaagct gaagatcttt cattgccaag tgacagcctg
      781 cctgatatac attaacatgt acctgagcat catattcctc gcttttgttt ccattgaccg
      841 ctgcctccaa ctgacacatt cttgtaaaat ctatagaatt caggagccag gattcgccaa
      901 gatgatttct acagtcgtgt ggctgatggt gcttctgatt atggtcccta acatgatgat
      961 tccaattaag gatatcaagg aaaagtctaa cgttggctgc atggagttta aaaaggaatt
     1021 tggtcgcaat tggcacctcc ttaccaactt catatgtgtt gccatctttc tgaatttctc
     1081 cgctatcatc ctcatcagca attgtctggt gatccgccag ctctacagaa ataaggacaa
     1141 cgaaaattat ccaaatgtga agaaggcgct gataaatata ctgttggtga ccactggata
     1201 tatcatttgt tttgtgccct atcacattgt gaggattcca tacacgctga gtcagacaga
     1261 ggtaattacc gactgttcaa cccgcatcag cttgttcaag gccaaagagg ctacactcct
     1321 tctggcagtc tctaacctgt gctttgaccc aatcctttat taccacctca gcaaggcttt
     1381 ccggagcaag gtaaccgaaa ctttcgccag tccaaaggag acaaaagcgc agaaagaaaa
     1441 gctccgatgt gaaaacaatg cgatcgatac cggtggacgc accggtgaat tcgatgaggc
     1501 cagcaccacc gtgtctaaga ccgagacttc tcaggtggcc cctgcctaac tcgagtctag
     1561 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     1621 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     1681 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     1741 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     1801 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     1861 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1921 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1981 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2041 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2101 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2161 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2221 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2281 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2341 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     2401 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2461 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2521 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     2581 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     2641 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     2701 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     2761 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     2821 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     2881 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     2941 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3001 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3061 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3121 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3181 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3241 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3301 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3361 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     3421 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3481 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     3541 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3601 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     3661 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3721 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3781 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     3841 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3901 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3961 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4021 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4081 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4141 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4201 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4261 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4321 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4381 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4441 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4501 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4561 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4621 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4681 gaacggtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4741 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4801 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4861 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4921 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4981 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5041 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5101 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5161 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5221 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5281 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5341 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5401 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5461 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5521 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5581 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5641 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5701 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5761 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5821 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5881 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5941 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     6001 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     6061 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     6121 caaaatttaa gctacaacaa ggcaaggctt gaccgacatt tgcatgaaga atctgcttag
     6181 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
     6241 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     6301 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     6361 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     6421 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     6481 tatgccaagt acgcccc
//
