LOCUS       sequence-450779-        6606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses his-tagged, 3C-cleavable 20D3Q mutant 2N4R tau for 
            recombinant protein production in bacteria .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6606)
  AUTHORS   Dickson JR, Sobolewski RGR, Fernandes AR, Cooper JM, Fan Z, Chung M,
            Donahue C, Oakley DH, Strickland DK, Hyman BT
  TITLE     Alzheimer disease-associated tau post-translational modification 
            mimics impact tau propagation and uptake.
  JOURNAL   J Neuropathol Exp Neurol. 2025 Jun 1;84(6):459-470. doi: 
            10.1093/jnen/nlaf007.
  PUBMED    39984820
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"; journalName: 
            "J Neuropathol Exp Neurol"; date: "2025-06-1- 1"; volume: "84"; 
            issue: "6"; pages: "459-470"
COMMENT     Sequence Label: pET28.His.3C.20D3Q.Tau2N4R
FEATURES             Location/Qualifiers
     source          1..6606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     282..301
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     385..404
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        385..403
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    404..428
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             443..465
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             478..495
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             496..519
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1855..1872
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1925..1943)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1939..1986
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2023..2478
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2110..2129)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2320..2341
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2570..3385)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2570..3385)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3293..3312
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3377..3396
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3507..4095
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3996..4015
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4249..4266
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4281..4420
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4346..4365)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4506..4528
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4522..4713)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5522..6604)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6566..6585
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(6605..6606,1..76))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
       61 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
      121 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
      181 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
      241 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
      301 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
      361 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
      421 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac catggaacat
      481 catcatcatc atcatctgga ggtgctgttc caaggtcctc atatggctga gccccgccag
      541 gagttcgaag tgatggaaga tcacgctggg acgtacgggt tgggggacag gaaagatcag
      601 gggggctacg atatgcacca agaccaagag ggtgacacgg acgctggcct gaaagaatct
      661 cccctgcaga cccccactga ggacggatct gaggaaccgg gctctgaaac ctctgatgct
      721 aaggatgatc cagatgcgga agatgtgaca gcacccttag tggatgaggg agctcccggc
      781 aagcaggctg ccgcgcagcc ccacacggag atcccagaag gaaccacagc tgaagaagca
      841 ggcattggag acgatcccga tctggaagac gaagctgctg gtcacgtgac ccaagctcgc
      901 atggtcagta aaagcaaaga cgggactgga agcgatgaca aaaaagccaa gggggctgat
      961 ggtaaaacga agatcgccac accgcgggga gcagcccctc caggccagaa gggccaggcc
     1021 aacgccacca ggattccagc aaaaaccccg cccgctccaa aggatccacc cagctctggt
     1081 gaacctccaa aatcagggga tcgcagcggc tacagcgatc ccggctcccc aggcactccc
     1141 ggcagccgct cccgcgatcc ggatcttcca gatccaccca cccgggagcc caagaaggtg
     1201 gcagtggtcc gtgatccacc caaggatccg gattccgcca agagccgcct gcagacagcc
     1261 cccgtgccca tgccagacct gaagaatgtc aagtccaaga tcggcgatga tgagaacctg
     1321 aagcaccagc cgggaggcgg gaaggtgcag ataattaata agcagctgga tcttagcaac
     1381 gtccagtcca agtgtggctc aaaggataat atcaaacacg tcccgggagg cggcagtgtg
     1441 caaatagtct acaaaccagt tgacctgagc aaggtgacct ccaagtgtgg ctcattaggc
     1501 aacatccatc ataaaccagg aggtggccag gtggaagtaa aatctgagca gcttgacttc
     1561 aaggacagag tccagtcgca gattgggtcc ctggacaata tcacccacgt ccctggcgga
     1621 ggaaataaaa agattgaaac ccacaagctg accttccgcg agaacgccaa agccaagaca
     1681 gaccacgggg cggagatcgt gtacaaggat ccagtggtgg atggggacga tgatccacgg
     1741 catctcagca atgtctcctc caccggcagc atcgacatgg tagactcgcc ccagctcgcc
     1801 acgctagctg acgaggtgtc tgcctccctg gccaagcagg gtttgtagct cgagcaccac
     1861 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     1921 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1981 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     2041 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2101 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2161 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2221 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2281 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2341 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2401 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     2461 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2521 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     2581 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     2641 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     2701 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     2761 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     2821 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     2881 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     2941 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     3001 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     3061 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     3121 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     3181 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     3241 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     3301 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     3361 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     3421 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3481 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3541 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3601 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3661 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3721 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3781 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3841 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3901 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3961 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4021 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4081 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4141 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4201 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4261 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4321 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4381 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4441 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4501 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4561 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     4621 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     4681 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     4741 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     4801 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4861 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4921 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4981 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     5041 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     5101 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     5161 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     5221 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     5281 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     5341 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     5401 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5461 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     5521 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5581 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     5641 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     5701 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     5761 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     5821 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     5881 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     5941 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     6001 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     6061 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     6121 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     6181 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     6241 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     6301 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     6361 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     6421 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     6481 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     6541 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     6601 tcacat
//
