LOCUS       sequence-423294-        8229 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human RFWD3. N-terminal NanoLuc 
            luciferase tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8229)
  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 8229)
  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-N_RFWD3:1-774
FEATURES             Location/Qualifiers
     source          1..8229
                     /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"
     CDS             1065..1577
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(3997..4015)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    4047..4168
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4058..4077)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4112..4131
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4413..4770
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4413..4604
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4436..4456)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4621..4756
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4801..5838
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             5817..5825
                     /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    5862..5910
                     /note="synthetic polyadenylation signal"
     promoter        6066..6170
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6171..6239,6240..7031)
                     /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(6389..6408)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(7189..7777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7269..7288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7893..8176
                     /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 ggctagcgct caccatggtc ttcacactcg
     1081 aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa gtccttgaac
     1141 agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg atccaaagga
     1201 ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag
     1261 gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg taccctgtgg
     1321 atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac ggggttacgc
     1381 cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa
     1441 agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca
     1501 accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt
     1561 gcgaacgcat tctggcgggc tcgagcggcg cgatcgcttc cgaattcatg gctcatgaag
     1621 caatggaata tgatgttcag gtgcagttaa atcatgccga acaacagcca gctcctgctg
     1681 gcatggccag cagccaaggg ggaccagccc tcctccagcc tgttcctgct gatgtggtca
     1741 gcagccaggg ggtaccatcc atcctccagc cagctcctgc tgaggtgatc agcagccaag
     1801 cgacaccacc cctgctccag cctgctccgc aactgtctgt tgacctgaca gaagtggagg
     1861 tcttgggaga agacactgtg gagaacatca atccaagaac ttcagaacaa cataggcagg
     1921 gatctgatgg taatcacacc atcccagcat cttcgttgca ttcaatgacc aacttcatca
     1981 gcggactgca gagacttcat ggcatgctgg aattcctgag accttcatct tcaaaccaca
     2041 gtgtagggcc aatgagaaca agaaggaggg tatctgcttc acggagggca agagccggag
     2101 ggtctcagag gacagacagt gccaggttga gagcaccatt ggatgcttac tttcaggtga
     2161 gcaggaccca gcctgacttg ccagctacca cttatgattc agagactagg aatcctgtat
     2221 ctgaagagtt gcaggtgtct agtagttctg attctgacag tgacagctct gcagagtatg
     2281 gaggggttgt tgaccaggca gaggaatctg gagctgtcat tttagaagag caactagcag
     2341 gtgtctcagc agagcaagaa gttacatgta tcgatggagg caagaccctc cccaaacagc
     2401 catctcccca gaagtctgag cctctgctac cttctgcttc tatggatgag gaagaagggg
     2461 acacttgtac aatatgtctg gaacagtgga ccaatgctgg ggaccaccgg ctctcagcat
     2521 tacgctgtgg gcatctcttt gggtataggt gcatttccac gtggcttaaa ggacaagtac
     2581 gaaaatgtcc ccagtgcaac aagaaagcca ggcacagtga cattgtcgtc ctttatgccc
     2641 gaaccctgag agctttggac actagtgaac aggagcgcat gaaaagttcc ctactgaagg
     2701 aacagatgct aaggaaacag gccgagttag aatcagcaca gtgccgactc caactgcagg
     2761 tcctcactga taagtgcact aggcttcaaa ggcgtgttca ggacttgcaa aaacttacgt
     2821 cacatcaaag tcagaattta cagcaaccca ggggctccca agcatgggtc ctgagctgct
     2881 caccctccag ccagggccag cacaagcaca agtaccactt ccaaaagacc ttcacagtat
     2941 ctcaggcagg aaactgccgg atcatggcat actgtgatgc tctgagctgc ctggtgatat
     3001 cacagccttc tcctcaggcc tcttttcttc caggctttgg tgttaagatg ttgagtactg
     3061 ccaacatgaa gagcagtcag tacattccga tgcatggcaa acagatccgt ggactggcgt
     3121 ttagcagtta cctcagaggc ttgctactct ctgcttccct agacaacact attaaactga
     3181 ccagcctgga gacaaatacc gtggtccaga cttataatgc tggacgtcct gtctggagct
     3241 gttgctggtg tcttgatgag gctaactaca tctatgctgg actggccaat ggttcaattc
     3301 tggtatatga cgtgcgaaac acgagcagtc atgtgcagga gttagtagct cagaaagcca
     3361 gatgcccact ggtctccctg tcatacatgc ccagagctgc ctcagctgca tttccatatg
     3421 gtggggtgct ggctggaacc ttggaggatg cttcattctg ggaacagaaa atggactttt
     3481 ctcattggcc tcatgtgctg cccttggagc cagggggctg catagacttt cagacagaga
     3541 acagctcccg gcactgtctt gtgacctaca ggcctgataa aaatcacacc accatacgaa
     3601 gtgtgctgat ggaaatgtcc taccgactgg atgacactgg aaatccaatc tgctcctgcc
     3661 agcctgtaca tacatttttt ggaggaccta cttgcaaact attgaccaaa aatgccattt
     3721 tccaaagccc agagaatgat ggcaacatcc tggtgtgtac tggggatgaa gcagcaaatt
     3781 ctgccctgct gtgggatgct gccagtggct cgttgctcca ggacctacag accgatcagc
     3841 ctgtgttgga catctgccca tttgaggtga accgtaacag ctacttggct accttaacag
     3901 agaagatggt ccacatctat aagtgggagt aataatctag atttgggccc aattcctgca
     3961 ggattttgcg gccgcttgct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     4021 cccttggccg cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac
     4081 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     4141 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     4201 ggttcagggg gagatgtggg aggttttttt aagcaagtaa aacctctaca aatgtggtaa
     4261 aatcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
     4321 gcatctgtgc ggtatttcac accgcatacg cggatctgcg cagcaccatg gcctgaaata
     4381 acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat
     4441 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4501 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     4561 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4621 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4681 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     4741 ggcttttttg gaggcctagg cttttgcaaa aagctcgatt cttctgacac tagcgccacc
     4801 atgaagaagc ccgaactcac cgctaccagc gttgaaaaat ttctcatcga gaagttcgac
     4861 agtgtgagcg acctgatgca gttgtcggag ggcgaagaga gccgagcctt cagcttcgat
     4921 gtcggcggac gcggctatgt actgcgggtg aatagctgcg ctgatggctt ctacaaagac
     4981 cgctacgtgt accgccactt cgccagcgct gcactaccca tccccgaagt gttggacatc
     5041 ggcgagttca gcgagagcct gacatactgc atcagtagac gcgcccaagg cgttactctc
     5101 caagacctcc ccgaaacaga gctgcctgct gtgttacagc ctgtcgccga agctatggat
     5161 gctattgccg ccgccgacct cagtcaaacc agcggcttcg gcccattcgg gccccaaggc
     5221 atcggccagt acacaacctg gcgggatttc atttgcgcca ttgctgatcc ccatgtctac
     5281 cactggcaga ccgtgatgga cgacaccgtg tccgccagcg tagctcaagc cctggacgaa
     5341 ctgatgctgt gggccgaaga ctgtcccgag gtgcgccacc tcgtccatgc cgacttcggc
     5401 agcaacaacg tcctgaccga caacggccgc atcaccgccg taatcgactg gtccgaagct
     5461 atgttcgggg acagtcagta cgaggtggcc aacatcttct tctggcggcc ctggctggct
     5521 tgcatggagc agcagactcg ctacttcgag cgccggcatc ccgagctggc cggcagccct
     5581 cgtctgcgag cctacatgct gcgcatcggc ctggatcagc tctaccagag cctcgtggac
     5641 ggcaacttcg acgatgctgc ctgggctcaa ggccgctgcg atgccatcgt ccgcagcggg
     5701 gccggcaccg tcggtcgcac acaaatcgct cgccggagcg cagccgtatg gaccgacggc
     5761 tgcgtcgagg tgctggccga cagcggcaac cgccggccca gtacacgacc gcgcgctaag
     5821 gaggtaggtc gagtttagac tctagccatc acgatggccg caataaaata tctttatttt
     5881 cattacatct gtgtgttggt tttttgtgtg aatcgatagc gataaggatc ctctttgcgc
     5941 ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt cagcaccgtt
     6001 tctgcggact ggctttctac gtaatggttt cttagacgtc aggtggcact tttcggggaa
     6061 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     6121 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     6181 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     6241 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     6301 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     6361 ttccaatgat gagcactttc aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     6421 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6481 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6541 ccataaccat gagtgataac actgcggcca acttacttct gacaactatc ggaggaccga
     6601 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6661 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6721 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6781 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6841 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6901 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6961 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     7021 agcattggta attcgaaatg accgaccaag cgacgcccaa ccggtatcag ctcactcaaa
     7081 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     7141 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     7201 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     7261 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     7321 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     7381 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     7441 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     7501 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     7561 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     7621 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     7681 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     7741 caagcagcag attacgcgca gaaaaaaagg atttcaagaa gatcctttga tcttttctac
     7801 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     7861 aaaaaggatc ttcacctaga tccttttata gtccggaaat acaggaacgc acgctggatg
     7921 gcccttcgct gggatggtga aaccatgaaa aatggcagct tcagtggatt aagtgggggt
     7981 aatgtggcct gtaccctctg gttgcatagg tattcatacg gttaaaattt atcaggcgcg
     8041 attgcggcag tttttcgggt ggtttgttgc catttttacc tgtctgctgc cgtgatcgcg
     8101 ctgaacgcgt tttagcggtg cgtacaatta agggattatg gtaaatccac ttactgtctg
     8161 ccctcgtagc catcgagata aaccgcagta ctccggccac gatgcgtccg gcgtagagga
     8221 tcgagatct
//
