LOCUS       sequence-430836-        6551 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 6551)
  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 6551)
  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: GPR77-DuET
FEATURES             Location/Qualifiers
     source          1..6551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     440..459
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     693..710
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    827..848
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(863..868,869..886,887..893)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    901..917
                     /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     906..928
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     925..941
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     925..941
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    990..1111
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1001..1020)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1055..1074
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1285..2079)
                     /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(1396..1415)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2006..2025
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2146..2475)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2160..2295
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2214..2233)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2460..2480
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2489..2917)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2599..2620)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2811..2830
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2963..3187
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3176..3193
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4453..4472)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4454..4472)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4517..4720)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4546..4566)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4721..5100
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5272..5291
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5366..5470
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5471..5539,5540..6331)
                     /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(5689..5708)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6502..6551,1..539)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
       61 taactggctt cagcagagcg cagataccaa ataccgttct tctagtgtag ccgtagttag
      121 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
      181 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
      241 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
      301 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
      361 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
      421 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
      481 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
      541 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
      601 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
      661 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
      721 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
      781 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      841 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      901 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct
      961 agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac
     1021 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     1081 tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat
     1141 ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt
     1201 catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg
     1261 tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
     1321 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
     1381 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
     1441 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
     1501 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag
     1561 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
     1621 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
     1681 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     1741 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     1801 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     1861 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
     1921 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     1981 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     2041 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     2101 ctcttgatca gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag
     2161 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc
     2221 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     2281 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     2341 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga
     2401 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     2461 ccctaactga cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca
     2521 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     2581 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     2641 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     2701 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     2761 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     2821 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     2881 acacccgccg cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag
     2941 ctggttcttt ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg
     3001 tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac
     3061 ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca
     3121 ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa
     3181 ggcacagtcg aggctgatca gcgggtttaa acgggccctc tagactcgag ttaggcaggg
     3241 gccacctgag aagtctcggt cttagacacg gtggtgctgg cctcatcgaa ttcaccggtg
     3301 cgtccaccgg tatcgatgac ttccatctcg gacaccaagt cgtgagatgt agacttttta
     3361 gagtccacac tttcatcctg tccctggctc tctcggaggg cccagtggca cgctgctggc
     3421 aggctcctcc ggagttgtgc ccgcccaaag taaaggaaca acatggggtt cagacaactg
     3481 tgggccaggg cgagtcccac aatcaacggc tcggcgcgca aagcccgagc cagaagggct
     3541 gagttgggag ctgccacagt cagtaccaag cccaacaagt ggtaaggggc ccagcacacg
     3601 aagaagccca ccacgatagc tgtgcccagc ggccgacagc gtcttgcggc ccagcagagc
     3661 aaggcgctgt ggcatgaagc aacggcaacc aggggaccca ggaatccaaa aagaaagcga
     3721 atggcggtca ctgcgttctc agtactggaa cttcccccat agtcaactac gcactgcagc
     3781 ctggctggga agtgctcttg gtgcagtctt ctgtatatgg cgctaggaac tgtcaacagc
     3841 agtgcgaggg tccacgcagc cccacaagcc acctggacgc cgcacgcgcg ttgcacggtg
     3901 ctccaccagg caggtccgag ggcgagaaag cacaagtcgg cagacaaggc agccaaaagg
     3961 agcacggagg catacattgt gagcagaatt atgctaggca atgccctgca gccaactgcg
     4021 ccgtacggcc aatgaccccc tcttgcgata ggcacggcca aaatgggcaa gctgagacag
     4081 cacaacaaat ctgccacggc aaggtgcaga agccacgttg cgcccactcg ccgtctcgcg
     4141 accttaccgg caacccaagc gaccatggca ttgcccggca cgccaaccag aaaaattgcg
     4201 gcatacaggg ggaggggggc caccctcagg gggtcgatgg ccagacaagc tccgtccagg
     4261 caatcgacag gccggtcgga caggtcagag tagtccccgt actcgtagct tacggaatca
     4321 ttgcccatat cgatgctggc gtcatcatcg tccttgtagt cggcgaatac caggcagaag
     4381 atgtagctca gggcgatgat cgtcttcatg gtggcgctaa gcttaagttt aaacgctagc
     4441 cagcttgggt ctccctatag tgagtcgtat taatttcgat aagccagtaa gcagtgggtt
     4501 ctctagttag ccagagagct ctgcttatat agacctccca ccgtacacgc ctaccgccca
     4561 tttgcgtcaa tggggcggag ttgttacgac attttggaaa gtcccgttga ttttggtgcc
     4621 aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt gagtcaaacc
     4681 gctatccacg cccattgatg tactgccaaa accgcatcac catggtaata gcgatgacta
     4741 atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc
     4801 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt
     4861 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
     4921 tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt
     4981 tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg
     5041 ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc
     5101 aacgcgtata tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat
     5161 tcttcatgca aatgtcggtc aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact
     5221 actcagcgac ctccaacaca caagcaggga gcagatactg gcttaactat gcggcatcag
     5281 agcagattgt actgagagtg caccataggg gatcgggaga tctcccgatc cgtcgacgtc
     5341 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5401 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5461 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5521 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5581 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5641 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5701 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5761 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5821 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5881 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5941 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6001 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6061 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6121 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6181 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6241 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6301 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6361 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6421 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6481 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6541 aacaaaaaaa c
//
