LOCUS       sequence-431809-        6656 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 6656)
  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 6656)
  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: GPR45-DuET
FEATURES             Location/Qualifiers
     source          1..6656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(78..97)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     688..707
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(828..1157)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      842..977
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(896..915)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1142..1162
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1171..1599)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1281..1302)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1493..1512
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1645..1869
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1858..1875
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(3240..3259)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3241..3259)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3304..3507)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3333..3353)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3508..3887
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4059..4078
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4153..4257
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4258..4326,4327..5118)
                     /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(4476..4495)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5289..5877
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5778..5797
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6031..6048
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6165..6186
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6201..6206,6207..6224,6225..6231)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6239..6255
                     /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     6244..6266
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6263..6279
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6263..6279
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    6328..6449
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6339..6358)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6393..6412
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(6623..6656,1..761))
                     /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"
ORIGIN
        1 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
       61 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
      121 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
      181 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
      241 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
      301 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
      361 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
      421 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
      481 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
      541 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
      601 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
      661 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
      721 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
      781 gtctcttgat cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct
      841 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg
      901 gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
      961 ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga
     1021 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct
     1081 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     1141 caccctaact gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct
     1201 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     1261 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     1321 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     1381 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     1441 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     1501 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     1561 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc
     1621 agctggttct ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat
     1681 tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac
     1741 acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg
     1801 caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag
     1861 aaggcacagt cgaggctgat cagcgggttt aaacgggccc tctagactcg agttaggcag
     1921 gggccacctg agaagtctcg gtcttagaca cggtggtgct ggcctcatcg aattcaccgg
     1981 tgcgtccacc ggtatcgatg acggcgctct gattttcgtt acagacgtat actgttgacg
     2041 gctgtatccg tcttctgatc ctctcgggta ctttaggaag aatctggaat gtttgaggca
     2101 ggagctcaat gcacgcttcc cggaatttct tgatccgcca acagtacact attggattaa
     2161 agacggactt caggtaggac agccagagga cgcaagttga ggtggcgtag aaagaggagc
     2221 cgcagtaaaa ccgttgtgaa aagacactca gcaatgagta gacgctgtgg gggagccaac
     2281 agaggctgaa tccaacgaag agaatcagaa tcgtggtaaa ggctttggtt ttgaaggaga
     2341 gatcgacgga aacttgctgc tgccgttgca accgtctaag tccagctcta gtgagctgcc
     2401 tcaggtccag agagtcgctt tgattatgca ctctaacggc gttcttcctc acggtattca
     2461 ggatgcacat ataggcacaa agcatcacgc cgaagggggc gaagaagacg gccacgacca
     2521 gagtcactac gtaggcgcgg tcagcgggca actcggtgta ccccagtaca cactggggag
     2581 ccctggctgg cacctccacg agggtccagc cggtcaaact aggaccagcg atacagaaag
     2641 aaagtaccca agagacagcg atgataacct ttgctctcct agggttcagt ttatcctgtc
     2701 tctggactat aatcaggaat cggtccacgc tgatgatcag caaaatggcg actccctcca
     2761 gcacgaaaaa ccagtaaagt gtagcagaga gcctgcaaaa atgatcacca aagtgccacc
     2821 tcacggttat cagtgtaact gccgtgaagg gcatacagca caaagacagc atgatgtcgc
     2881 taaaggcaag cgtagccaaa agcaggttaa tcgcagaccg cattgcaggc ctttgataaa
     2941 caattataca cactacagta tttccaagaa accccaccac ggtcatcagc agcatgacta
     3001 tagccaggga gatccggagg ggagcgggca gttgagtact tccactgtcg gatgcgttgc
     3061 tcgtgttaag cagcagatag gtataggcct caagtgatgt agagttgcat gccatatcga
     3121 tgctggcgtc atcatcgtcc ttgtagtcgg cgaataccag gcagaagatg tagctcaggg
     3181 cgatgatcgt cttcatggtg ccgctaagct taagtttaaa cgctagccag cttgggtctc
     3241 cctatagtga gtcgtattaa tttcgataag ccagtaagca gtgggttctc tagttagcca
     3301 gagagctctg cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg
     3361 ggcggagttg ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc
     3421 attgacgtca atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc
     3481 attgatgtac tgccaaaacc gcatcaccat ggtaatagcg atgactaata cgtagatgta
     3541 ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt
     3601 accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa
     3661 gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt
     3721 tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc
     3781 aggcgggcca tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa
     3841 tgaccccgta attgattact attaataact agtcaataat caatgtcaac gcgtatatct
     3901 ggcccgtaca tcgcgaagca gcgcaaaacg cctaacccta agcagattct tcatgcaaat
     3961 gtcggtcaag ccttgccttg ttgtagctta aattttgctc gcgcactact cagcgacctc
     4021 caacacacaa gcagggagca gatactggct taactatgcg gcatcagagc agattgtact
     4081 gagagtgcac cataggggat cgggagatct cccgatccgt cgacgtcagg tggcactttt
     4141 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4201 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4261 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4321 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4381 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4441 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4501 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4561 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4621 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4681 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4741 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4801 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4861 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4921 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4981 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5041 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5101 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5161 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5221 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5281 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5341 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5401 actggcttca gcagagcgca gataccaaat accgttcttc tagtgtagcc gtagttaggc
     5461 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5521 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5581 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5641 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5701 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5761 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5821 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5881 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5941 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     6001 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     6061 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     6121 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     6181 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     6241 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagctctag
     6301 ctagaggtcg acggtataca gacatgataa gatacattga tgagtttgga caaaccacaa
     6361 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     6421 taaccattat aagctgcaat aaacaagttg gggtgggcga agaactccag catgagatcc
     6481 ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca
     6541 tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc
     6601 atttcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcg
//
