LOCUS       sequence-329856-        7268 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of N. furzeri DDX5-Delta-PrD(1-535) mutant in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7268)
  AUTHORS   Itamar Harel , Yiwen R. Chen, Inbal Ziv, Param Priya Singh, Paloma 
            Navarro Negredo, Uri Goshtchevsky, Wei Wang, Gwendoline Astre, Eitan
            Moses, Andrew McKay, Ben E. Machado, Katja Hebestreit, Sifei Yin, 
            View Alejandro Sanchez Alvarado, Daniel F. Jarosz, Anne Brunet
  TITLE     Identification of protein aggregates in the aging vertebrate brain 
            with prion-like and phase separation properties
  JOURNAL   BioRxiv
REFERENCE   2  (bases 1 to 7268)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BioRxiv"
COMMENT     Sequence Label: pDEST17-His10-Smt3-DDX5-Delta-PrD(kf)-SNAP
FEATURES             Location/Qualifiers
     source          1..7268
                     /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(2133..2157)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2159..2182
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2201..2746
                     /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(2814..2832)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2828..2875
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3241..3246,3247..3263,3264..3269))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3296..3314)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3382..3486
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3487..3555,3556..4347)
                     /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(3705..3724)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4518..5106
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5007..5026
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5260..5277
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5292..5431
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5357..5376)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5517..5539
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5533..5724)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     7186..7205
                     /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 cgacccagct ttcttgtaca aagtggttga
     2161 ctacaaggat gacgatgaca agcttaccgg tgagttcgct atggacaaag attgcgaaat
     2221 gaaacgtacc accctggata gcccgctggg caaactggaa ctgagcggct gcgaacaggg
     2281 cctgcatgaa attaaactgc tgggtaaagg caccagcgcg gccgatgcgg ttgaagttcc
     2341 ggccccggcc gccgtgctgg gtggtccgga accgctgatg caggcgaccg cgtggctgaa
     2401 cgcgtatttt catcagccgg aagcgattga agaatttccg gttccggcgc tgcatcatcc
     2461 ggtgtttcag caggagagct ttacccgtca ggtgctgtgg aaactgctga aagtggttaa
     2521 atttggcgaa gtgattagct atcagcagct ggcggccctg gcgggtaatc cggcggccac
     2581 cgccgccgtt aaaaccgcgc tgagcggtaa cccggtgccg attctgattc cgtgccatcg
     2641 tgtggttagc tctagcggtg cggttggcgg ttatgaaggt ggtctggcgg tgaaagagtg
     2701 gctgctggcc catgaaggtc atcgtctggg taaaccgggt ctgggacctg caggctgata
     2761 aggattcgag gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     2821 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     2881 aggaggaact atatccggat atccacagga cgggtgtggt cgccatgatc gcgtagtcga
     2941 tagtggctcc aagtagcgaa gcgagcagga ctgggcggcg gccaaagcgg tcggacagtg
     3001 ctccgagaac gggtgcgcat agaaattgca tcaacgcata tagcgctagc agcacgccat
     3061 agtgactggc gatgctgtcg gaatggacga tatcccgcaa gaggcccggc agtaccggca
     3121 taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg atgagcgcat
     3181 tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc
     3241 attaaagctt atcgatgata agctgtcaaa catgagaatt cttgaagacg aaagggcctc
     3301 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     3361 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3421 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3481 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3541 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3601 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3661 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3721 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3781 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3841 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3901 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     3961 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4021 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4081 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4141 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4201 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4261 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4321 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4381 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4441 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4501 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4561 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4621 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4681 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4741 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4801 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4861 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4921 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4981 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5041 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5101 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5161 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5221 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5281 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5341 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     5401 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     5461 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5521 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     5581 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     5641 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     5701 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     5761 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     5821 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     5881 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     5941 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     6001 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     6061 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     6121 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     6181 gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat
     6241 ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa
     6301 gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc
     6361 attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg
     6421 tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa
     6481 atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat
     6541 ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg
     6601 ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc
     6661 gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc
     6721 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     6781 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     6841 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     6901 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     6961 ttgaaggctc tcaagggcat cggtcgatcg acgctctccc ttatgcgact cctgcattag
     7021 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     7081 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     7141 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     7201 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     7261 gaggatcg
//
