LOCUS       sequence-423369-        7239 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human RBBP4. C-terminal NanoLuc 
            luciferase tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7239)
  AUTHORS   Ackloo S, Li F, Szewczyk M, Seitova A, Loppnau P, Zeng H, Xu J, 
            Ahmad S, Arnautova YA, Baghaie AJ, Beldar S, Bolotokova A, Centrella
            PA, Chau I, Clark MA, Cuozzo JW, Dehghani-Tafti S, Disch JS, Dong A,
            Dumas A, Feng JA, Ghiabi P, Gibson E, Gilmer J, Goldman B, Green SR,
            Guie MA, Guilinger JP, Harms N, Herasymenko O, Houliston S, 
            Hutchinson A, Kearnes S, Keefe AD, Kimani SW, Kramer T, Kutera M, 
            Kwak HA, Lento C, Li Y, Liu J, Loup J, Machado RAC, Mulhern CJ, 
            Perveen S, Righetto GL, Riley P, Shrestha S, Sigel EA, Silva M, 
            Sintchak MD, Slakman BL, Taylor RD, Thompson J, Torng W, 
            Underkoffler C, von Rechenberg M, Walsh RT, Watson I, Wilson DJ, 
            Wolf E, Yadav M, Yazdi AK, Zhang J, Zhang Y, Santhakumar V, Edwards 
            AM, Barsyte-Lovejoy D, Schapira M, Brown PJ, Halabelian L, 
            Arrowsmith CH
  TITLE     A Target Class Ligandability Evaluation of WD40 Repeat-Containing 
            Proteins.
  JOURNAL   J Med Chem. 2025 Jan 23;68(2):1092-1112. doi: 
            10.1021/acs.jmedchem.4c02010. Epub 2024 Nov 4.
  PUBMED    39495097
REFERENCE   2  (bases 1 to 7239)
  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"; doi: 
            "10.1021/acs.jmedchem.4c02010"; journalName: "J Med Chem"; date: 
            "2025-01-23- 23"; volume: "68"; issue: "2"; pages: "1092-1112"
COMMENT     Sequence Label: pNLF1-C_RBBP4:1-425
FEATURES             Location/Qualifiers
     source          1..7239
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1033..1052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1079..1088
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2414..2926
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(3002..3020)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3057..3178
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3068..3087)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3122..3141
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3423..3780
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3423..3614
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3446..3466)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3631..3766
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3811..4848
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4827..4835
                     /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)"
     polyA_signal    4872..4920
                     /note="synthetic polyadenylation signal"
     promoter        5076..5180
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5181..5249,5250..6041)
                     /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(5399..5418)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6199..6787)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6279..6298)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6903..7186
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcaaa gcgatcgctt ccgaattcgc
     1081 caccatggcc gacaaggaag cagccttcga cgacgcagtg gaagaacgag tgatcaacga
     1141 ggaatacaaa atatggaaaa agaacacccc ttttctttat gatttggtga tgacccatgc
     1201 tctggagtgg cccagcctaa ctgcccagtg gcttccagat gtaaccagac cagaagggaa
     1261 agatttcagc attcatcgac ttgtcctggg gacacacaca tcggatgaac aaaaccatct
     1321 tgttatagcc agtgtgcagc tccctaatga tgatgctcag tttgatgcgt cacactacga
     1381 cagtgagaaa ggagaatttg gaggttttgg ttcagttagt ggaaaaattg aaatagaaat
     1441 caagatcaac catgaaggag aagtaaacag ggcccgttat atgccccaga acccttgtat
     1501 catcgcaaca aagactcctt ccagtgatgt tcttgtcttt gactatacaa aacatccttc
     1561 taaaccagat ccttctggag agtgcaaccc agacttgcgt ctccgtggac atcagaagga
     1621 aggctatggg ctttcttgga acccaaatct cagtgggcac ttacttagtg cttcagatga
     1681 ccataccatc tgcctgtggg acatcagtgc cgttccaaag gagggaaaag tggtagatgc
     1741 gaagaccatc tttacagggc atacggcagt agtagaagat gtttcctggc atctactcca
     1801 tgagtctctg tttgggtcag ttgctgatga tcagaaactt atgatttggg atactcgttc
     1861 aaacaatact tccaaaccaa gccactcagt tgatgctcac actgctgaag tgaactgcct
     1921 ttctttcaat ccttatagtg agttcattct tgccacagga tcagctgaca agactgttgc
     1981 cttgtgggat ctgagaaatc tgaaacttaa gttgcattcc tttgagtcac ataaggatga
     2041 aatattccag gttcagtggt cacctcacaa tgagactatt ttagcttcca gtggtactga
     2101 tcgcagactg aatgtctggg atttaagtaa aattggagag gaacaatccc cagaagatgc
     2161 agaagacggg ccaccagagt tgttgtttat tcatggtggt catactgcca agatatctga
     2221 tttctcctgg aatcccaatg aaccttgggt gatttgttct gtatcagaag acaatatcat
     2281 gcaagtgtgg caaatggcag agaacattta taatgatgaa gaccctgaag gaagcgtgga
     2341 tccagaagga caagggtccc ggatatctag atttgggccc aattcctgca ggcgagctct
     2401 cggctcgagc ggcgtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg
     2461 ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg
     2521 ggtgtccgta actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga
     2581 catccatgtc atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa
     2641 aatttttaag gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg
     2701 cacactggta atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga
     2761 aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa
     2821 caaaattatc gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat
     2881 caacggagtg accggctggc ggctgtgcga acgcattctg gcgtaaggcc gcgactctag
     2941 ggcatgcaag ctgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     3001 accgctgagc aataactagc ataacccctt ggggcggccg cttcgagcag acatgataag
     3061 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     3121 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     3181 caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggttttttt
     3241 aagcaagtaa aacctctaca aatgtggtaa aatcgaattt taacaaaata ttaacgctta
     3301 caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     3361 cggatctgcg cagcaccatg gcctgaaata acctctgaaa gaggaacttg gttaggtacc
     3421 ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3481 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3541 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3601 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3661 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3721 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3781 aagctcgatt cttctgacac tagcgccacc atgaagaagc ccgaactcac cgctaccagc
     3841 gttgaaaaat ttctcatcga gaagttcgac agtgtgagcg acctgatgca gttgtcggag
     3901 ggcgaagaga gccgagcctt cagcttcgat gtcggcggac gcggctatgt actgcgggtg
     3961 aatagctgcg ctgatggctt ctacaaagac cgctacgtgt accgccactt cgccagcgct
     4021 gcactaccca tccccgaagt gttggacatc ggcgagttca gcgagagcct gacatactgc
     4081 atcagtagac gcgcccaagg cgttactctc caagacctcc ccgaaacaga gctgcctgct
     4141 gtgttacagc ctgtcgccga agctatggat gctattgccg ccgccgacct cagtcaaacc
     4201 agcggcttcg gcccattcgg gccccaaggc atcggccagt acacaacctg gcgggatttc
     4261 atttgcgcca ttgctgatcc ccatgtctac cactggcaga ccgtgatgga cgacaccgtg
     4321 tccgccagcg tagctcaagc cctggacgaa ctgatgctgt gggccgaaga ctgtcccgag
     4381 gtgcgccacc tcgtccatgc cgacttcggc agcaacaacg tcctgaccga caacggccgc
     4441 atcaccgccg taatcgactg gtccgaagct atgttcgggg acagtcagta cgaggtggcc
     4501 aacatcttct tctggcggcc ctggctggct tgcatggagc agcagactcg ctacttcgag
     4561 cgccggcatc ccgagctggc cggcagccct cgtctgcgag cctacatgct gcgcatcggc
     4621 ctggatcagc tctaccagag cctcgtggac ggcaacttcg acgatgctgc ctgggctcaa
     4681 ggccgctgcg atgccatcgt ccgcagcggg gccggcaccg tcggtcgcac acaaatcgct
     4741 cgccggagcg cagccgtatg gaccgacggc tgcgtcgagg tgctggccga cagcggcaac
     4801 cgccggccca gtacacgacc gcgcgctaag gaggtaggtc gagtttagac tctagccatc
     4861 acgatggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg
     4921 aatcgatagc gataaggatc ctctttgcgc ttgcgttttc ccttgtccag atagcccagt
     4981 agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac gtaatggttt
     5041 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5101 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5161 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5221 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5281 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5341 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttc aaagttctgc
     5401 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5461 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5521 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5581 acttacttct gacaactatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5641 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5701 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5761 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5821 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5881 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5941 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6001 agatcgctga gataggtgcc tcactgatta agcattggta attcgaaatg accgaccaag
     6061 cgacgcccaa ccggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     6121 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     6181 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     6241 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     6301 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6361 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6421 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6481 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6541 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6601 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     6661 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6721 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6781 atttcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6841 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttata
     6901 gtccggaaat acaggaacgc acgctggatg gcccttcgct gggatggtga aaccatgaaa
     6961 aatggcagct tcagtggatt aagtgggggt aatgtggcct gtaccctctg gttgcatagg
     7021 tattcatacg gttaaaattt atcaggcgcg attgcggcag tttttcgggt ggtttgttgc
     7081 catttttacc tgtctgctgc cgtgatcgcg ctgaacgcgt tttagcggtg cgtacaatta
     7141 agggattatg gtaaatccac ttactgtctg ccctcgtagc catcgagata aaccgcagta
     7201 ctccggccac gatgcgtccg gcgtagagga tcgagatct
//
