LOCUS       sequence-329854-        7565 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of N. furzeri DDX5 in E. coli .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7565)
  AUTHORS   Harel I, Chen YR, Ziv I, Singh PP, Heinzer D, Navarro Negredo P, 
            Goshtchevsky U, Wang W, Astre G, Moses E, McKay A, Machado BE, 
            Hebestreit K, Yin S, Sanchez Alvarado A, Jarosz DF, Brunet A
  TITLE     Identification of protein aggregates in the aging vertebrate brain 
            with prion-like and phase-separation properties.
  JOURNAL   Cell Rep. 2024 Jun 25;43(6):112787. doi: 
            10.1016/j.celrep.2023.112787. Epub 2024 May 28.
  PUBMED    38810650
REFERENCE   2  (bases 1 to 7565)
  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"; journalName: "Cell Rep.";
            date: "2024-06-25"; volume: "43(6):112787. doi"; pages: " 
            10.1016/j.celrep.2023.112787. Epub 2024 May 28"
COMMENT     Sequence Label: pDEST17-His10-Smt3-DDX5(kf)-SNAP
FEATURES             Location/Qualifiers
     source          1..7565
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             71..93
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             113..142
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             155..166
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             179..472
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     protein_bind    485..509
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2430..2454)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2456..2479
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2498..3043
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     primer_bind     complement(3111..3129)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3125..3172
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3538..3543,3544..3560,3561..3566))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3593..3611)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3679..3783
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3784..3852,3853..4644)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4002..4021)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4815..5403
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5304..5323
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5557..5574
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5589..5728
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5654..5673)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5814..5836
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5830..6021)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     7483..7502
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc
       61 tagaaataat tttgtttaac tttaagaagg agatatacat atgtcgtact accatcacca
      121 tcaccatcac catcatcatc acagcagcgg ccatatcgaa ggtcgtcata tggctagcat
      181 gtcggactca gaagtcaatc aagaagctaa gccagaggtc aagccagaag tcaagcctga
      241 gactcacatc aatttaaagg tgtccgatgg atcttcagag atcttcttca agatcaaaaa
      301 gaccactcct ttaagaaggc tgatggaagc gttcgctaaa agacagggta aggaaatgga
      361 ctccttaaga ttcttgtacg acggtattag aattcaagct gatcagaccc ctgaagattt
      421 ggacatggag gataacgata ttattgaggc tcacagagaa cagattggtg gatccctcga
      481 atcaacaagt ttgtacaaaa aagcaggctt cgaaggagat aacaaaatgc ctggatttgc
      541 agacagagac cgcggcagag atagagggta tgggggtggc ccccctcggt ttggtggcgg
      601 tgggggagga ggcggcaaca gaggaggacc tctgctggga aagtttggaa accctggtga
      661 gaggctgcgc aagaagcact ggaaccttga tgaactacca aaatttcaaa agaatttcta
      721 ccaggaacac ccagatgtca cacacagacc actgcaggag gttgaccagt accgcagaaa
      781 caaagaagtt acagttaaag gcagagattg tcccaaaccg attgtaaaat tccatgaagc
      841 tgcttttccc aactacgtca tggatgtcat tgtcaaacag aactggactg aaccaactcc
      901 gatccagtct caggggtggc ccattgtcct gagtggaaaa gacatggtcg gcatcgctca
      961 aacggggtct gggaagactt tggcatatct tctgcctgca gtcgttcaca tccaacatca
     1021 gcccttcttg gagcacggag acgggcctat ttgcttggtg ctggcaccaa cccgtgagct
     1081 ggctcagcag gtgcaacagg tggctgctga gtacggcagg gcctcccgtc tcaagagtac
     1141 atgcatctat ggtggtgctc ccaaaggacc acaaatccgg gaccttgaga ggggtgttga
     1201 gatttgcatc gccaccccag gtcgcctcat tgatttcctg gaatgtggta aaactaattt
     1261 gcgtcgttgc acctatctag tgctggatga agctgaccgc atgctggaca tgggctttga
     1321 acctcagatc cgcaagatag tggatcaaat acggccagac cgtcagactc tgatgtggag
     1381 tgccacctgg cctaaagaag ttcgtcagct ggctgaggac ttcttgaagg attacgtcca
     1441 gatcaacatt ggcgccctgc agctcagcgc caatcataac atcctgcaaa tagtagatgt
     1501 ttgcaatgac ttagaaaagg aagacaaact gattcggttg ctggaggaga tcatgagtga
     1561 aaaggaaaac aagaccatca tttttgtgga gaccaaaagg cgttgtgatg agctcaccag
     1621 gaggatgaga agagacggat ggcctgcaat gggaatccat ggagataaga gccagcagga
     1681 gagggactgg gtccttaacg agttcagatt tggcaaagct ccgattctca ttgctacaga
     1741 cgtggcctcc cggggcttag atgtggagga tgtgaaattt gtcatcaatt atgactaccc
     1801 taactcctct gaggattata tccaccgcat tggacgcaca gcccgaagtc aaaaaacggg
     1861 cacggcctac accttcttca cccccaacaa catgaaacag gcaggtgacc tgatctctgt
     1921 gctccgtgaa gccaaccagg ccatcaaccc caaactgatc cagatggctg aggacagagg
     1981 aggtcgcggt agggggggaa gaggtggcta caaggatgac cgtcgcgata ggtattctgg
     2041 gggtgggagg agtaattttg gcggtagtgg ctttagagac agggaccatg acagaggttt
     2101 cggcaatggg ccgaagacca catttggtgg cagcaaggcc caaaatagtg gcaattatgg
     2161 tggcaacagt ggtaactctt gtggtagcta tggcaacaat aactacaaca acagtaatgg
     2221 acagggtagt tttagtggtc cacctaacca aggtggcttt gggaaccagg gcttccaagc
     2281 ccctcctcaa tttggtggca tgcagagggc tgcacagaat ggtgttaatc atccaccatt
     2341 tccttttaac gctcagccac ccccgccaca gaaccagcag ccccctccac caatggtgcc
     2401 ctacccaatt ccaccaccgt ttccacaaga cccagctttc ttgtacaaag tggttgacta
     2461 caaggatgac gatgacaagc ttaccggtga gttcgctatg gacaaagatt gcgaaatgaa
     2521 acgtaccacc ctggatagcc cgctgggcaa actggaactg agcggctgcg aacagggcct
     2581 gcatgaaatt aaactgctgg gtaaaggcac cagcgcggcc gatgcggttg aagttccggc
     2641 cccggccgcc gtgctgggtg gtccggaacc gctgatgcag gcgaccgcgt ggctgaacgc
     2701 gtattttcat cagccggaag cgattgaaga atttccggtt ccggcgctgc atcatccggt
     2761 gtttcagcag gagagcttta cccgtcaggt gctgtggaaa ctgctgaaag tggttaaatt
     2821 tggcgaagtg attagctatc agcagctggc ggccctggcg ggtaatccgg cggccaccgc
     2881 cgccgttaaa accgcgctga gcggtaaccc ggtgccgatt ctgattccgt gccatcgtgt
     2941 ggttagctct agcggtgcgg ttggcggtta tgaaggtggt ctggcggtga aagagtggct
     3001 gctggcccat gaaggtcatc gtctgggtaa accgggtctg ggacctgcag gctgataagg
     3061 attcgaggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     3121 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     3181 aggaactata tccggatatc cacaggacgg gtgtggtcgc catgatcgcg tagtcgatag
     3241 tggctccaag tagcgaagcg agcaggactg ggcggcggcc aaagcggtcg gacagtgctc
     3301 cgagaacggg tgcgcataga aattgcatca acgcatatag cgctagcagc acgccatagt
     3361 gactggcgat gctgtcggaa tggacgatat cccgcaagag gcccggcagt accggcataa
     3421 ccaagcctat gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt
     3481 tagatttcat acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt
     3541 aaagcttatc gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg
     3601 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     3661 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3721 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3781 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     3841 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3901 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3961 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4021 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4081 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4141 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4201 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4261 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4321 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4381 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4441 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4501 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4561 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4621 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4681 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4741 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4801 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4861 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4921 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     4981 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5041 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5101 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5161 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5221 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5281 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5341 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5401 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5461 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5521 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5581 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     5641 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     5701 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     5761 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     5821 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     5881 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     5941 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     6001 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     6061 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     6121 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     6181 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     6241 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     6301 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     6361 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     6421 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     6481 cacccgtggc caggacccaa cgctgcccga gatgcgccgc gtgcggctgc tggagatggc
     6541 ggacgcgatg gatatgttct gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa
     6601 ttgattggct ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt
     6661 caggtcgagg tggcccggct ccatgcaccg cgacgcaacg cggggaggca gacaaggtat
     6721 agggcggcgc ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc
     6781 gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg taagagccgc gagcgatcct
     6841 tgaagctgtc cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcgggc
     6901 atcccgatgc cgccggaagc gagaagaatc ataatgggga aggccatcca gcctcgcgtc
     6961 gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca tgccggcgat aatggcctgc
     7021 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     7081 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     7141 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     7201 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     7261 aaggctctca agggcatcgg tcgatcgacg ctctccctta tgcgactcct gcattaggaa
     7321 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     7381 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     7441 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     7501 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     7561 gatcg
//
