LOCUS       sequence-425965-        6650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human RFWD3. C-terminal HiBiT 
            tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6650)
  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 6650)
  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-C_RFWD3:1-774
FEATURES             Location/Qualifiers
     source          1..6650
                     /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    3713..3834
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3724..3743)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3949..3982)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3992..4039
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4071..4865
                     /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(4125..4144)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4735..4754
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(5023..5611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5103..5122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5737..5785
                     /note="synthetic polyadenylation signal"
     CDS             complement(5809..6207)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6204..6213
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6238..6595)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6252..6387
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6404..6595
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6552..6572
                     /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 ataggggcga tcgcaacaag cttcgggaag
     1201 atctggagct agcggcgggg gatccggtgg gaattcgaca ccatggctca tgaagcaatg
     1261 gaatatgatg ttcaggtgca gttaaatcat gccgaacaac agccagctcc tgctggcatg
     1321 gccagcagcc aagggggacc agccctcctc cagcctgttc ctgctgatgt ggtcagcagc
     1381 cagggggtac catccatcct ccagccagct cctgctgagg tgatcagcag ccaagcgaca
     1441 ccacccctgc tccagcctgc tccgcaactg tctgttgacc tgacagaagt ggaggtcttg
     1501 ggagaagaca ctgtggagaa catcaatcca agaacttcag aacaacatag gcagggatct
     1561 gatggtaatc acaccatccc agcatcttcg ttgcattcaa tgaccaactt catcagcgga
     1621 ctgcagagac ttcatggcat gctggaattc ctgagacctt catcttcaaa ccacagtgta
     1681 gggccaatga gaacaagaag gagggtatct gcttcacgga gggcaagagc cggagggtct
     1741 cagaggacag acagtgccag gttgagagca ccattggatg cttactttca ggtgagcagg
     1801 acccagcctg acttgccagc taccacttat gattcagaga ctaggaatcc tgtatctgaa
     1861 gagttgcagg tgtctagtag ttctgattct gacagtgaca gctctgcaga gtatggaggg
     1921 gttgttgacc aggcagagga atctggagct gtcattttag aagagcaact agcaggtgtc
     1981 tcagcagagc aagaagttac atgtatcgat ggaggcaaga ccctccccaa acagccatct
     2041 ccccagaagt ctgagcctct gctaccttct gcttctatgg atgaggaaga aggggacact
     2101 tgtacaatat gtctggaaca gtggaccaat gctggggacc accggctctc agcattacgc
     2161 tgtgggcatc tctttgggta taggtgcatt tccacgtggc ttaaaggaca agtacgaaaa
     2221 tgtccccagt gcaacaagaa agccaggcac agtgacattg tcgtccttta tgcccgaacc
     2281 ctgagagctt tggacactag tgaacaggag cgcatgaaaa gttccctact gaaggaacag
     2341 atgctaagga aacaggccga gttagaatca gcacagtgcc gactccaact gcaggtcctc
     2401 actgataagt gcactaggct tcaaaggcgt gttcaggact tgcaaaaact tacgtcacat
     2461 caaagtcaga atttacagca acccaggggc tcccaagcat gggtcctgag ctgctcaccc
     2521 tccagccagg gccagcacaa gcacaagtac cacttccaaa agaccttcac agtatctcag
     2581 gcaggaaact gccggatcat ggcatactgt gatgctctga gctgcctggt gatatcacag
     2641 ccttctcctc aggcctcttt tcttccaggc tttggtgtta agatgttgag tactgccaac
     2701 atgaagagca gtcagtacat tccgatgcat ggcaaacaga tccgtggact ggcgtttagc
     2761 agttacctca gaggcttgct actctctgct tccctagaca acactattaa actgaccagc
     2821 ctggagacaa ataccgtggt ccagacttat aatgctggac gtcctgtctg gagctgttgc
     2881 tggtgtcttg atgaggctaa ctacatctat gctggactgg ccaatggttc aattctggta
     2941 tatgacgtgc gaaacacgag cagtcatgtg caggagttag tagctcagaa agccagatgc
     3001 ccactggtct ccctgtcata catgcccaga gctgcctcag ctgcatttcc atatggtggg
     3061 gtgctggctg gaaccttgga ggatgcttca ttctgggaac agaaaatgga cttttctcat
     3121 tggcctcatg tgctgccctt ggagccaggg ggctgcatag actttcagac agagaacagc
     3181 tcccggcact gtcttgtgac ctacaggcct gataaaaatc acaccaccat acgaagtgtg
     3241 ctgatggaaa tgtcctaccg actggatgac actggaaatc caatctgctc ctgccagcct
     3301 gtacatacat tttttggagg acctacttgc aaactattga ccaaaaatgc cattttccaa
     3361 agcccagaga atgatggcaa catcctggtg tgtactgggg atgaagcagc aaattctgcc
     3421 ctgctgtggg atgctgccag tggctcgttg ctccaggacc tacagaccga tcagcctgtg
     3481 ttggacatct gcccatttga ggtgaaccgt aacagctact tggctacctt aacagagaag
     3541 atggtccaca tctataagtg ggagggctcg agcggtggga gctccggtgt gagcggctgg
     3601 cggctgttca agaagattag ctaagtttaa acggccgcga ctctagactg caggcatgca
     3661 agctgatttg gctgctgcca ccgctgagca aggccgcttc gagcagacat gataagatac
     3721 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     3781 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     3841 aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt ttttttaagc
     3901 aagtaaaacc tctacaaatg tggtaaaatc ttaattaaga cttctaatat aacttcgtat
     3961 agcatacatt atacgaagtt atggttccac tgttgacaat taatcatcgg catagtatat
     4021 cggcatagta taatacgaca aggtgaggaa ctaaacccag gaggcagatc atgattgaac
     4081 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     4141 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     4201 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     4261 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     4321 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     4381 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     4441 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4501 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4561 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     4621 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4681 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4741 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     4801 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4861 tctgattcga aatgaccgac caagcgacgc ccaaccggta tcagctcact caaaggcggt
     4921 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4981 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5041 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5101 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5161 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5221 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5281 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5341 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5401 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5461 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5521 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5581 gcagattacg cgcagaaaaa aaggatttca agaagatcct ttgatctttt ctacggggtc
     5641 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5701 gatcttcacc tagatccttt tatagtccgg aatcgtcaca caaaaaacca acacacagat
     5761 gtaatgaaaa taaagatatt ttattgcggc catcgtgatg gctagagttt agccctccca
     5821 cacataacca gagggcagca attcacgaat cccaactgcc gtcggctgtc catcactgtc
     5881 cttcactatg gctttgatcc caggatgcag atcgagaagc acctgtcggc accgtccgca
     5941 ggggctcaag atgcccctgt tctcatttcc gatcgcgacg atacaagtca ggttgccagc
     6001 tgccgcagca gcagcagtgc ccagcaccac gagttctgca caaggtcccc cagtaaaatg
     6061 atatacattg acaccagtga agatgcggcc gtcgctagag agagctgcgc tggcgacgct
     6121 gtagtcttca gagatgggga tgctgttgat tgtagccgtt gctctttcaa tgagggtgga
     6181 ttcttcttga gacaaaggct tggccatggt ggcgctagtg tcagaagaat cgagcttttt
     6241 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga
     6301 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
     6361 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
     6421 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     6481 cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
     6541 ggactttcca caccctaact gacacacatt ccacagctgg ttctttccgc ctcagaaggt
     6601 acctaaccaa gttcctcttt cagaggttat ttcaggccat ggtgctgcgc
//
