LOCUS       sequence-425987-        6606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human RFWD3. N-terminal HiBiT 
            tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6606)
  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 6606)
  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: pBiT3.1-N_RFWD3:1-774
FEATURES             Location/Qualifiers
     source          1..6606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        338..641
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        642..845
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     796..816
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          981..1113
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1096..1115
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1157..1176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1157..1175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3669..3790
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3680..3699)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3905..3938)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3948..3995
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4027..4821
                     /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(4081..4100)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4691..4710
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4979..5567)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5059..5078)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5693..5741
                     /note="synthetic polyadenylation signal"
     CDS             complement(5765..6163)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6160..6169
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6194..6551)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6208..6343
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6360..6551
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6508..6528
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 aagacgcgtt aattaagacg tcggcagtga aaaaaatgct ttatttgtga aatttgtgat
       61 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
      121 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
      181 ctctacaaat gtggtatggc tgattatgat ccgcggccgc aatacgtcga cgttatcagc
      241 tgacttcgta cgagagccta ggattatggc gcgccactag ttattaatag taatcaatta
      301 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      361 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      421 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      481 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca
      541 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta
      601 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      661 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      721 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata
      781 accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      841 gagctggttt agtgaaccgt cagatcacta gaatctttat tgcggtagtt tatcacagtt
      901 aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc tgcagaagtt
      961 ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat
     1021 agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc
     1081 ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccagttc aattacagct
     1141 cttaaggcta gagtattaat acgactcact ataggggtta gcgatcgcca tggtgagcgg
     1201 ctggcggctg ttcaagaaga ttagcgggag ctccggtggc tcgagcggta tggctcatga
     1261 agcaatggaa tatgatgttc aggtgcagtt aaatcatgcc gaacaacagc cagctcctgc
     1321 tggcatggcc agcagccaag ggggaccagc cctcctccag cctgttcctg ctgatgtggt
     1381 cagcagccag ggggtaccat ccatcctcca gccagctcct gctgaggtga tcagcagcca
     1441 agcgacacca cccctgctcc agcctgctcc gcaactgtct gttgacctga cagaagtgga
     1501 ggtcttggga gaagacactg tggagaacat caatccaaga acttcagaac aacataggca
     1561 gggatctgat ggtaatcaca ccatcccagc atcttcgttg cattcaatga ccaacttcat
     1621 cagcggactg cagagacttc atggcatgct ggaattcctg agaccttcat cttcaaacca
     1681 cagtgtaggg ccaatgagaa caagaaggag ggtatctgct tcacggaggg caagagccgg
     1741 agggtctcag aggacagaca gtgccaggtt gagagcacca ttggatgctt actttcaggt
     1801 gagcaggacc cagcctgact tgccagctac cacttatgat tcagagacta ggaatcctgt
     1861 atctgaagag ttgcaggtgt ctagtagttc tgattctgac agtgacagct ctgcagagta
     1921 tggaggggtt gttgaccagg cagaggaatc tggagctgtc attttagaag agcaactagc
     1981 aggtgtctca gcagagcaag aagttacatg tatcgatgga ggcaagaccc tccccaaaca
     2041 gccatctccc cagaagtctg agcctctgct accttctgct tctatggatg aggaagaagg
     2101 ggacacttgt acaatatgtc tggaacagtg gaccaatgct ggggaccacc ggctctcagc
     2161 attacgctgt gggcatctct ttgggtatag gtgcatttcc acgtggctta aaggacaagt
     2221 acgaaaatgt ccccagtgca acaagaaagc caggcacagt gacattgtcg tcctttatgc
     2281 ccgaaccctg agagctttgg acactagtga acaggagcgc atgaaaagtt ccctactgaa
     2341 ggaacagatg ctaaggaaac aggccgagtt agaatcagca cagtgccgac tccaactgca
     2401 ggtcctcact gataagtgca ctaggcttca aaggcgtgtt caggacttgc aaaaacttac
     2461 gtcacatcaa agtcagaatt tacagcaacc caggggctcc caagcatggg tcctgagctg
     2521 ctcaccctcc agccagggcc agcacaagca caagtaccac ttccaaaaga ccttcacagt
     2581 atctcaggca ggaaactgcc ggatcatggc atactgtgat gctctgagct gcctggtgat
     2641 atcacagcct tctcctcagg cctcttttct tccaggcttt ggtgttaaga tgttgagtac
     2701 tgccaacatg aagagcagtc agtacattcc gatgcatggc aaacagatcc gtggactggc
     2761 gtttagcagt tacctcagag gcttgctact ctctgcttcc ctagacaaca ctattaaact
     2821 gaccagcctg gagacaaata ccgtggtcca gacttataat gctggacgtc ctgtctggag
     2881 ctgttgctgg tgtcttgatg aggctaacta catctatgct ggactggcca atggttcaat
     2941 tctggtatat gacgtgcgaa acacgagcag tcatgtgcag gagttagtag ctcagaaagc
     3001 cagatgccca ctggtctccc tgtcatacat gcccagagct gcctcagctg catttccata
     3061 tggtggggtg ctggctggaa ccttggagga tgcttcattc tgggaacaga aaatggactt
     3121 ttctcattgg cctcatgtgc tgcccttgga gccagggggc tgcatagact ttcagacaga
     3181 gaacagctcc cggcactgtc ttgtgaccta caggcctgat aaaaatcaca ccaccatacg
     3241 aagtgtgctg atggaaatgt cctaccgact ggatgacact ggaaatccaa tctgctcctg
     3301 ccagcctgta catacatttt ttggaggacc tacttgcaaa ctattgacca aaaatgccat
     3361 tttccaaagc ccagagaatg atggcaacat cctggtgtgt actggggatg aagcagcaaa
     3421 ttctgccctg ctgtgggatg ctgccagtgg ctcgttgctc caggacctac agaccgatca
     3481 gcctgtgttg gacatctgcc catttgaggt gaaccgtaac agctacttgg ctaccttaac
     3541 agagaagatg gtccacatct ataagtggga gtaatctaga ggtcaagctt cgggggttta
     3601 aacctgcagg catgcaagct gatttggctg ctgccaccgc tgagcaaggc cgcttcgagc
     3661 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     3721 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3781 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg
     3841 ggaggttttt ttaagcaagt aaaacctcta caaatgtggt aaaatcttaa ttaagacttc
     3901 taatataact tcgtatagca tacattatac gaagttatgg ttccactgtt gacaattaat
     3961 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa acccaggagg
     4021 cagatcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4081 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4141 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4201 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4261 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4321 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4381 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4441 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4501 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4561 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4621 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4681 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4741 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4801 cttcttgacg agttcttctg attcgaaatg accgaccaag cgacgcccaa ccggtatcag
     4861 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4921 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4981 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5041 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5101 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5161 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5221 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5281 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5341 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5401 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5461 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5521 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atttcaagaa gatcctttga
     5581 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5641 tgagattatc aaaaaggatc ttcacctaga tccttttata gtccggaatc gtcacacaaa
     5701 aaaccaacac acagatgtaa tgaaaataaa gatattttat tgcggccatc gtgatggcta
     5761 gagtttagcc ctcccacaca taaccagagg gcagcaattc acgaatccca actgccgtcg
     5821 gctgtccatc actgtccttc actatggctt tgatcccagg atgcagatcg agaagcacct
     5881 gtcggcaccg tccgcagggg ctcaagatgc ccctgttctc atttccgatc gcgacgatac
     5941 aagtcaggtt gccagctgcc gcagcagcag cagtgcccag caccacgagt tctgcacaag
     6001 gtcccccagt aaaatgatat acattgacac cagtgaagat gcggccgtcg ctagagagag
     6061 ctgcgctggc gacgctgtag tcttcagaga tggggatgct gttgattgta gccgttgctc
     6121 tttcaatgag ggtggattct tcttgagaca aaggcttggc catggtggcg ctagtgtcag
     6181 aagaatcgag ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga
     6241 atagctcaga ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat
     6301 ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg
     6361 ggactatggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     6421 ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc
     6481 ctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac agctggttct
     6541 ttccgcctca gaaggtacct aaccaagttc ctctttcaga ggttatttca ggccatggtg
     6601 ctgcgc
//
