LOCUS       sequence-423295-        8280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human RFWD3. C-terminal NanoLuc 
            luciferase tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8280)
  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 8280)
  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_RFWD3:1-774
FEATURES             Location/Qualifiers
     source          1..8280
                     /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             3455..3967
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(4043..4061)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    4098..4219
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4109..4128)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4163..4182
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4464..4821
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4464..4655
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4487..4507)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4672..4807
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4852..5889
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             5868..5876
                     /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    5913..5961
                     /note="synthetic polyadenylation signal"
     promoter        6117..6221
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6222..6290,6291..7082)
                     /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(6440..6459)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(7240..7828)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7320..7339)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7944..8227
                     /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 caccatggct catgaagcaa tggaatatga tgttcaggtg cagttaaatc atgccgaaca
     1141 acagccagct cctgctggca tggccagcag ccaaggggga ccagccctcc tccagcctgt
     1201 tcctgctgat gtggtcagca gccagggggt accatccatc ctccagccag ctcctgctga
     1261 ggtgatcagc agccaagcga caccacccct gctccagcct gctccgcaac tgtctgttga
     1321 cctgacagaa gtggaggtct tgggagaaga cactgtggag aacatcaatc caagaacttc
     1381 agaacaacat aggcagggat ctgatggtaa tcacaccatc ccagcatctt cgttgcattc
     1441 aatgaccaac ttcatcagcg gactgcagag acttcatggc atgctggaat tcctgagacc
     1501 ttcatcttca aaccacagtg tagggccaat gagaacaaga aggagggtat ctgcttcacg
     1561 gagggcaaga gccggagggt ctcagaggac agacagtgcc aggttgagag caccattgga
     1621 tgcttacttt caggtgagca ggacccagcc tgacttgcca gctaccactt atgattcaga
     1681 gactaggaat cctgtatctg aagagttgca ggtgtctagt agttctgatt ctgacagtga
     1741 cagctctgca gagtatggag gggttgttga ccaggcagag gaatctggag ctgtcatttt
     1801 agaagagcaa ctagcaggtg tctcagcaga gcaagaagtt acatgtatcg atggaggcaa
     1861 gaccctcccc aaacagccat ctccccagaa gtctgagcct ctgctacctt ctgcttctat
     1921 ggatgaggaa gaaggggaca cttgtacaat atgtctggaa cagtggacca atgctgggga
     1981 ccaccggctc tcagcattac gctgtgggca tctctttggg tataggtgca tttccacgtg
     2041 gcttaaagga caagtacgaa aatgtcccca gtgcaacaag aaagccaggc acagtgacat
     2101 tgtcgtcctt tatgcccgaa ccctgagagc tttggacact agtgaacagg agcgcatgaa
     2161 aagttcccta ctgaaggaac agatgctaag gaaacaggcc gagttagaat cagcacagtg
     2221 ccgactccaa ctgcaggtcc tcactgataa gtgcactagg cttcaaaggc gtgttcagga
     2281 cttgcaaaaa cttacgtcac atcaaagtca gaatttacag caacccaggg gctcccaagc
     2341 atgggtcctg agctgctcac cctccagcca gggccagcac aagcacaagt accacttcca
     2401 aaagaccttc acagtatctc aggcaggaaa ctgccggatc atggcatact gtgatgctct
     2461 gagctgcctg gtgatatcac agccttctcc tcaggcctct tttcttccag gctttggtgt
     2521 taagatgttg agtactgcca acatgaagag cagtcagtac attccgatgc atggcaaaca
     2581 gatccgtgga ctggcgttta gcagttacct cagaggcttg ctactctctg cttccctaga
     2641 caacactatt aaactgacca gcctggagac aaataccgtg gtccagactt ataatgctgg
     2701 acgtcctgtc tggagctgtt gctggtgtct tgatgaggct aactacatct atgctggact
     2761 ggccaatggt tcaattctgg tatatgacgt gcgaaacacg agcagtcatg tgcaggagtt
     2821 agtagctcag aaagccagat gcccactggt ctccctgtca tacatgccca gagctgcctc
     2881 agctgcattt ccatatggtg gggtgctggc tggaaccttg gaggatgctt cattctggga
     2941 acagaaaatg gacttttctc attggcctca tgtgctgccc ttggagccag ggggctgcat
     3001 agactttcag acagagaaca gctcccggca ctgtcttgtg acctacaggc ctgataaaaa
     3061 tcacaccacc atacgaagtg tgctgatgga aatgtcctac cgactggatg acactggaaa
     3121 tccaatctgc tcctgccagc ctgtacatac attttttgga ggacctactt gcaaactatt
     3181 gaccaaaaat gccattttcc aaagcccaga gaatgatggc aacatcctgg tgtgtactgg
     3241 ggatgaagca gcaaattctg ccctgctgtg ggatgctgcc agtggctcgt tgctccagga
     3301 cctacagacc gatcagcctg tgttggacat ctgcccattt gaggtgaacc gtaacagcta
     3361 cttggctacc ttaacagaga agatggtcca catctataag tgggagtcta gatttgggcc
     3421 caattcctgc aggcgagctc tcggctcgag cggcgtcttc acactcgaag atttcgttgg
     3481 ggactggcga cagacagccg gctacaacct ggaccaagtc cttgaacagg gaggtgtgtc
     3541 cagtttgttt cagaatctcg gggtgtccgt aactccgatc caaaggattg tcctgagcgg
     3601 tgaaaatggg ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcggcga
     3661 ccaaatgggc cagatcgaaa aaatttttaa ggtggtgtac cctgtggatg atcatcactt
     3721 taaggtgatc ctgcactatg gcacactggt aatcgacggg gttacgccga acatgatcga
     3781 ctatttcgga cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac
     3841 agggaccctg tggaacggca acaaaattat cgacgagcgc ctgatcaacc ccgacggctc
     3901 cctgctgttc cgagtaacca tcaacggagt gaccggctgg cggctgtgcg aacgcattct
     3961 ggcgtaaggc cgcgactcta gggcatgcaa gctgatccgg ctgctaacaa agcccgaaag
     4021 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcggcc
     4081 gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     4141 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     4201 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     4261 ggagatgtgg gaggtttttt taagcaagta aaacctctac aaatgtggta aaatcgaatt
     4321 ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg
     4381 cggtatttca caccgcatac gcggatctgc gcagcaccat ggcctgaaat aacctctgaa
     4441 agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     4501 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4561 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4621 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4681 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4741 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     4801 ggaggcctag gcttttgcaa aaagctcgat tcttctgaca ctagcgccac catgaagaag
     4861 cccgaactca ccgctaccag cgttgaaaaa tttctcatcg agaagttcga cagtgtgagc
     4921 gacctgatgc agttgtcgga gggcgaagag agccgagcct tcagcttcga tgtcggcgga
     4981 cgcggctatg tactgcgggt gaatagctgc gctgatggct tctacaaaga ccgctacgtg
     5041 taccgccact tcgccagcgc tgcactaccc atccccgaag tgttggacat cggcgagttc
     5101 agcgagagcc tgacatactg catcagtaga cgcgcccaag gcgttactct ccaagacctc
     5161 cccgaaacag agctgcctgc tgtgttacag cctgtcgccg aagctatgga tgctattgcc
     5221 gccgccgacc tcagtcaaac cagcggcttc ggcccattcg ggccccaagg catcggccag
     5281 tacacaacct ggcgggattt catttgcgcc attgctgatc cccatgtcta ccactggcag
     5341 accgtgatgg acgacaccgt gtccgccagc gtagctcaag ccctggacga actgatgctg
     5401 tgggccgaag actgtcccga ggtgcgccac ctcgtccatg ccgacttcgg cagcaacaac
     5461 gtcctgaccg acaacggccg catcaccgcc gtaatcgact ggtccgaagc tatgttcggg
     5521 gacagtcagt acgaggtggc caacatcttc ttctggcggc cctggctggc ttgcatggag
     5581 cagcagactc gctacttcga gcgccggcat cccgagctgg ccggcagccc tcgtctgcga
     5641 gcctacatgc tgcgcatcgg cctggatcag ctctaccaga gcctcgtgga cggcaacttc
     5701 gacgatgctg cctgggctca aggccgctgc gatgccatcg tccgcagcgg ggccggcacc
     5761 gtcggtcgca cacaaatcgc tcgccggagc gcagccgtat ggaccgacgg ctgcgtcgag
     5821 gtgctggccg acagcggcaa ccgccggccc agtacacgac cgcgcgctaa ggaggtaggt
     5881 cgagtttaga ctctagccat cacgatggcc gcaataaaat atctttattt tcattacatc
     5941 tgtgtgttgg ttttttgtgt gaatcgatag cgataaggat cctctttgcg cttgcgtttt
     6001 cccttgtcca gatagcccag tagctgacat tcatccgggg tcagcaccgt ttctgcggac
     6061 tggctttcta cgtaatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     6121 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     6181 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     6241 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     6301 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     6361 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     6421 tgagcacttt caaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     6481 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     6541 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     6601 tgagtgataa cactgcggcc aacttacttc tgacaactat cggaggaccg aaggagctaa
     6661 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6721 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6781 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6841 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6901 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6961 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     7021 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     7081 aattcgaaat gaccgaccaa gcgacgccca accggtatca gctcactcaa aggcggtaat
     7141 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     7201 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     7261 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     7321 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     7381 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     7441 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     7501 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     7561 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     7621 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     7681 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     7741 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     7801 gattacgcgc agaaaaaaag gatttcaaga agatcctttg atcttttcta cggggtctga
     7861 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     7921 cttcacctag atccttttat agtccggaaa tacaggaacg cacgctggat ggcccttcgc
     7981 tgggatggtg aaaccatgaa aaatggcagc ttcagtggat taagtggggg taatgtggcc
     8041 tgtaccctct ggttgcatag gtattcatac ggttaaaatt tatcaggcgc gattgcggca
     8101 gtttttcggg tggtttgttg ccatttttac ctgtctgctg ccgtgatcgc gctgaacgcg
     8161 ttttagcggt gcgtacaatt aagggattat ggtaaatcca cttactgtct gccctcgtag
     8221 ccatcgagat aaaccgcagt actccggcca cgatgcgtcc ggcgtagagg atcgagatct
//
