LOCUS       sequence-430876-        6653 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 6653)
  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 6653)
  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: NPS-DuET
FEATURES             Location/Qualifiers
     source          1..6653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      105..693
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     594..613
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     847..864
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    981..1002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1017..1022,1023..1040,1041..1047)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1055..1071
                     /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     1060..1082
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1079..1095
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1079..1095
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1144..1265
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1155..1174)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1209..1228
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1439..2233)
                     /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(1550..1569)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2160..2179
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2300..2629)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2314..2449
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2368..2387)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2614..2634
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2643..3071)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2753..2774)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2965..2984
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3117..3341
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3330..3347
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4709..4728)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4710..4728)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4773..4976)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4802..4822)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4977..5356
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5528..5547
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5622..5726
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5727..5795,5796..6587)
                     /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(5945..5964)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
       61 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
      121 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
      181 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
      241 ccaaataccg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
      301 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
      361 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
      421 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
      481 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
      541 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
      601 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
      661 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
      721 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
      781 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
      841 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
      901 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
      961 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     1021 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     1081 ggaaacagct atgaccatga ttacgccaag ctctagctag aggtcgacgg tatacagaca
     1141 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     1201 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     1261 aagttggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat catccagccg
     1321 gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt ggaatcgaaa
     1381 tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt cgaaccccag agtcccgctc
     1441 agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac
     1501 cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg
     1561 tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc
     1621 cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga
     1681 cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg gctggcgcga
     1741 gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac
     1801 gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg
     1861 tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag
     1921 atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag
     1981 tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg
     2041 ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg
     2101 ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg
     2161 cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat
     2221 cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagatccg aaaatggata
     2281 tacaagctcc cgggagcttt ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact
     2341 tctggaatag ctcagaggca gaggcggcct cggcctctgc ataaataaaa aaaattagtc
     2401 agccatgggg cggagaatgg gcggaactgg gcggagttag gggcgggatg ggcggagtta
     2461 ggggcgggac tatggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
     2521 gggagcctgg ggactttcca cacctggttg ctgactaatt gagatgcatg ctttgcatac
     2581 ttctgcctgc tggggagcct ggggactttc cacaccctaa ctgacacaca ttccacagaa
     2641 ttaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     2701 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     2761 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     2821 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     2881 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     2941 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3001 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3061 acagggcgcg tggggatacc ccctagagcc ccagctggtt ctttccgcct cagaagccat
     3121 agagcccacc gcatccccag catgcctgct attgtcttcc caatcctccc ccttgctgtc
     3181 ctgccccacc ccacccccca gaatagaatg acacctactc agacaatgcg atgcaatttc
     3241 ctcattttat taggaaagga cagtgggagt ggcaccttcc agggtcaagg aaggcacggg
     3301 ggaggggcaa acaacagatg gctggcaact agaaggcaca gtcgaggctg atcagcgggt
     3361 ttaaacgggc cctctagact cgagttaggc aggggccacc tgagaagtct cggtcttaga
     3421 cacggtggtg ctggcctcat cgaattcacc ggtgcgtcca ccggtatcga tgatgaattc
     3481 gggctttgac agaatctgca tctcatgcct ttcagtcctc tcgcggaatg tcatcctgga
     3541 gtcctggctc ctctgctctc tacatggaaa ggaaatggag gaactaaaga cacaatatat
     3601 cagagggtta atcgcactat tgagggcggg caggttctgg ataatcacgc tggcgtaaaa
     3661 tcgctcttgc gtgtcaggca acaaattgaa attgtccagg atatcgaaga gaaagtaggg
     3721 actccagcag cagatgaaag ccaggataat aataatggaa tacttaatgg ccttgatctt
     3781 agccttagag atgagaccgc ggttgtagct gctgcaaagc ttcccatctg agcaattgct
     3841 aatcacggtt tcgtaagtct tagattttat ccagatggtg cgaatgacaa tcccgtacat
     3901 tatgctgatg atcgtcagtg gaatgaagta taccaggaaa gccacgatcg tcatgtaagg
     3961 ggtccagtaa ctgtcgtctg gccaaagggc ccagcattgc acctctccat ttgaaagtgt
     4021 tcttttgccg aaaatgatca gtgtgggaat actgaacagg aaggacaagg accaggcaat
     4081 aactatgagg actctggcct gcttctcgcc ttgcaggaac ttcataggat agacgatagc
     4141 atgatatcgg tcgattgaca gtgacaccag gacataggta ctagcataca ggaggacaac
     4201 ctgcagatac cgaaccactc tgcacacaag atccggtgct gtgaaatccc ctgtgaacct
     4261 ccagttgatg tcggtcagaa tgttcaccag tccggtgaaa ctgtcagtta ttgcgagctg
     4321 agtaacaaag aaggtcattc gactcttctt cttccggcgc caagtgctaa acaagaccac
     4381 actgttgcca acaatagtga aaacgaacaa gacccacagg gttatcagct gttctgtctt
     4441 gaaggaatag tagaatgagc cccattcctt accttccacc acctcggtaa atgtgacggt
     4501 ctcggtacaa gcgacagggc tggagtccag ggtctggccg gtacctgatg agtcgaagga
     4561 tccctcggta aaatttgcag gcatatcgat gctggcgtca tcatcgtcct tgtagtcggc
     4621 gaataccagg cagaagatgt agctcagggc gatgatcgtc ttcatggtgg cgctaagctt
     4681 aagtttaaac gctagccagc ttgggtctcc ctatagtgag tcgtattaat ttcgataagc
     4741 cagtaagcag tgggttctct agttagccag agagctctgc ttatatagac ctcccaccgt
     4801 acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt tggaaagtcc
     4861 cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag acttggaaat
     4921 ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg catcaccatg
     4981 gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca taaggtcatg
     5041 tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt
     5101 ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat
     5161 tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat tattgacgtc
     5221 aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg
     5281 cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta
     5341 gtcaataatc aatgtcaacg cgtatatctg gcccgtacat cgcgaagcag cgcaaaacgc
     5401 ctaaccctaa gcagattctt catgcaaatg tcggtcaagc cttgccttgt tgtagcttaa
     5461 attttgctcg cgcactactc agcgacctcc aacacacaag cagggagcag atactggctt
     5521 aactatgcgg catcagagca gattgtactg agagtgcacc ataggggatc gggagatctc
     5581 ccgatccgtc gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5641 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5701 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5761 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5821 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5881 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5941 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     6001 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6061 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6121 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6181 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6241 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6301 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6361 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6421 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6481 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6541 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6601 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa agg
//
