LOCUS       sequence-450808-        6639 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses his-tagged, 3C-cleavable, 86b (split luciferase 
            fragment)-tagged 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 6639)
  TITLE     Hyman lab Tau2N4R plasmids
REFERENCE   2  (bases 1 to 6639)
  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"
COMMENT     Sequence Label: pET28.His.3C.86b.20D3Q.Tau2N4R
FEATURES             Location/Qualifiers
     source          1..6639
                     /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             1888..1905
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1958..1976)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1972..2019
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2056..2511
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2143..2162)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2353..2374
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2603..3418)
                     /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(2603..3418)
                     /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     3326..3345
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3410..3429
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3540..4128
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4029..4048
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4282..4299
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4314..4453
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4379..4398)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4539..4561
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4555..4746)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5555..6637)
                     /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     6599..6618
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(6638..6639,1..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 caaggtcctg tgagtggctg gcgactgttc
      541 aagaagatca gccatatggc tgagccccgc caggagttcg aagtgatgga agatcacgct
      601 gggacgtacg ggttggggga caggaaagat caggggggct acgatatgca ccaagaccaa
      661 gagggtgaca cggacgctgg cctgaaagaa tctcccctgc agacccccac tgaggacgga
      721 tctgaggaac cgggctctga aacctctgat gctaaggatg atccagatgc ggaagatgtg
      781 acagcaccct tagtggatga gggagctccc ggcaagcagg ctgccgcgca gccccacacg
      841 gagatcccag aaggaaccac agctgaagaa gcaggcattg gagacgatcc cgatctggaa
      901 gacgaagctg ctggtcacgt gacccaagct cgcatggtca gtaaaagcaa agacgggact
      961 ggaagcgatg acaaaaaagc caagggggct gatggtaaaa cgaagatcgc cacaccgcgg
     1021 ggagcagccc ctccaggcca gaagggccag gccaacgcca ccaggattcc agcaaaaacc
     1081 ccgcccgctc caaaggatcc acccagctct ggtgaacctc caaaatcagg ggatcgcagc
     1141 ggctacagcg atcccggctc cccaggcact cccggcagcc gctcccgcga tccggatctt
     1201 ccagatccac ccacccggga gcccaagaag gtggcagtgg tccgtgatcc acccaaggat
     1261 ccggattccg ccaagagccg cctgcagaca gcccccgtgc ccatgccaga cctgaagaat
     1321 gtcaagtcca agatcggcga tgatgagaac ctgaagcacc agccgggagg cgggaaggtg
     1381 cagataatta ataagcagct ggatcttagc aacgtccagt ccaagtgtgg ctcaaaggat
     1441 aatatcaaac acgtcccggg aggcggcagt gtgcaaatag tctacaaacc agttgacctg
     1501 agcaaggtga cctccaagtg tggctcatta ggcaacatcc atcataaacc aggaggtggc
     1561 caggtggaag taaaatctga gcagcttgac ttcaaggaca gagtccagtc gcagattggg
     1621 tccctggaca atatcaccca cgtccctggc ggaggaaata aaaagattga aacccacaag
     1681 ctgaccttcc gcgagaacgc caaagccaag acagaccacg gggcggagat cgtgtacaag
     1741 gatccagtgg tggatgggga cgatgatcca cggcatctca gcaatgtctc ctccaccggc
     1801 agcatcgaca tggtagactc gccccagctc gccacgctag ctgacgaggt gtctgcctcc
     1861 ctggccaagc agggtttgta gctcgagcac caccaccacc accactgaga tccggctgct
     1921 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1981 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     2041 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2101 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2161 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2221 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2281 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2341 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2401 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2461 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     2521 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2581 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     2641 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     2701 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     2761 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     2821 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     2881 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     2941 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     3001 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3061 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     3121 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3181 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3241 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3301 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3361 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3421 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     3481 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3541 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3601 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3661 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3721 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3781 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3841 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3901 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3961 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4021 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4081 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4141 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4201 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4261 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4321 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4381 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4441 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4501 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4561 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     4621 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     4681 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     4741 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     4801 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     4861 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     4921 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     4981 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     5041 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     5101 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     5161 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     5221 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     5281 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     5341 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     5401 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     5461 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     5521 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5581 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5641 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     5701 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     5761 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     5821 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     5881 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     5941 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     6001 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     6061 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     6121 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     6181 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     6241 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     6301 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     6361 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     6421 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     6481 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     6541 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     6601 catactctgc gacatcgtat aacgttactg gtttcacat
//
