LOCUS       sequence-450770-        8269 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses tau propagation ORF with V5 tag on T403D mutant 2N4R tau.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8269)
  TITLE     Hyman lab Tau2N4R plasmids
REFERENCE   2  (bases 1 to 8269)
  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: pcDNA3.1(-).TagRFP.T-2A-V5-T403D.Tau2N4R-WPRE
FEATURES             Location/Qualifiers
     source          1..8269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    921..973
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      981..990
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             987..1718
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     CDS             1728..1781
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1791..1832
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    3178..3766
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3231..3251)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3649..3660)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3860..3877)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3866..4090
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4136..4564
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4223..4242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4433..4454
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4573..4593)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4578..4907
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4758..4893
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4820..4839
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4974..5768
                     /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(5028..5047)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5638..5657
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5942..6063
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5979..5998)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6033..6052
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6112..6128)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6112..6128)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6125..6147)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6136..6152
                     /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)."
     promoter        complement(join(6160..6166,6167..6184,6185..6190))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6205..6226
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6343..6360)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6514..7102)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6594..6613)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7273..8064,8065..8133))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7896..7915
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8134..8238)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaacgg gccctctaga ctcgagctca agcttcgaat tctgcagtcg acggtaccgc
      961 gggcccggga tccaccggtc gccaccatgg tgtctaaggg cgaagagctg attaaggaga
     1021 acatgcacat gaagctgtac atggagggca ccgtgaacaa ccaccacttc aagtgcacat
     1081 ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag gtggtcgagg
     1141 gcggccctct ccccttcgcc ttcgacatcc tggctaccag cttcatgtac ggcagcagaa
     1201 ccttcatcaa ccacacccag ggcatccccg acttctttaa gcagtccttc cctgagggct
     1261 tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca
     1321 ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcccat
     1381 ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccaacacc gagatgctgt
     1441 accccgctga cggcggcctg gaaggcagaa ccgacatggc cctgaagctc gtgggcgggg
     1501 gccacctgat ctgcaacttc aagaccacat acagatccaa gaaacccgct aagaacctca
     1561 agatgcccgg cgtctactat gtggaccaca gactggaaag aatcaaggag gccgacaaag
     1621 agacctacgt cgagcagcac gaggtggctg tggccagata ctgcgacctc cctagcaaac
     1681 tggggcacaa acttaatggc atggacgagc tgtacaaggg taccggcgag ggcagaggaa
     1741 gtctgctaac atgcggtgac gtcgaggaga atcctggccc aaccggtaaa ggtaagccta
     1801 tccctaaccc tctcctcggt ctcgattcta cgactagtgc tgagccccgc caggagttcg
     1861 aagtgatgga agatcacgct gggacgtacg ggttggggga caggaaagat caggggggct
     1921 acaccatgca ccaagaccaa gagggtgaca cggacgctgg cctgaaagaa tctcccctgc
     1981 agacccccac tgaggacgga tctgaggaac cgggctctga aacctctgat gctaagagca
     2041 ctccaacagc ggaagatgtg acagcaccct tagtggatga gggagctccc ggcaagcagg
     2101 ctgccgcgca gccccacacg gagatcccag aaggaaccac agctgaagaa gcaggcattg
     2161 gagacacccc cagcctggaa gacgaagctg ctggtcacgt gacccaagct cgcatggtca
     2221 gtaaaagcaa agacgggact ggaagcgatg acaaaaaagc caagggggct gatggtaaaa
     2281 cgaagatcgc cacaccgcgg ggagcagccc ctccaggcca gaagggccag gccaacgcca
     2341 ccaggattcc agcaaaaacc ccgcccgctc caaagacacc acccagctct ggtgaacctc
     2401 caaaatcagg ggatcgcagc ggctacagca gccccggctc cccaggcact cccggcagcc
     2461 gctcccgcac cccgtccctt ccaaccccac ccacccggga gcccaagaag gtggcagtgg
     2521 tccgtactcc acccaagtcg ccgtcttccg ccaagagccg cctgcagaca gcccccgtgc
     2581 ccatgccaga cctgaagaat gtcaagtcca agatcggctc cactgagaac ctgaagcacc
     2641 agccgggagg cgggaaggtg cagataatta ataagaagct ggatcttagc aacgtccagt
     2701 ccaagtgtgg ctcaaaggat aatatcaaac acgtcccggg aggcggcagt gtgcaaatag
     2761 tctacaaacc agttgacctg agcaaggtga cctccaagtg tggctcatta ggcaacatcc
     2821 atcataaacc aggaggtggc caggtggaag taaaatctga gaagcttgac ttcaaggaca
     2881 gagtccagtc gaagattggg tccctggaca atatcaccca cgtccctggc ggaggaaata
     2941 aaaagattga aacccacaag ctgaccttcc gcgagaacgc caaagccaag acagaccacg
     3001 gggcggagat cgtgtacaag tcgccagtgg tgtctgggga cgattctcca cggcatctca
     3061 gcaatgtctc ctccaccggc agcatcgaca tggtagactc gccccagctc gccacgctag
     3121 ctgacgaggt gtctgcctcc ctggccaagc agggtttgta gggatccgat tatcgataat
     3181 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     3241 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     3301 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     3361 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3421 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     3481 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3541 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     3601 tgtgttgcca cctggattct gcgcgggacg cccttctgct acgtcccttc ggccctcaat
     3661 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3721 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
     3781 gcgatatctg cagaattcca ccacactgga ctagtggatc cgagctcggt accaagctta
     3841 agtttaaacc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     3901 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3961 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     4021 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     4081 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     4141 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4201 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4261 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4321 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     4381 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4441 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     4501 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4561 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     4621 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     4681 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     4741 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     4801 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     4861 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     4921 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     4981 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     5041 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     5101 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     5161 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     5221 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     5281 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     5341 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     5401 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     5461 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     5521 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     5581 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     5641 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     5701 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     5761 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     5821 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     5881 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     5941 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     6001 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     6061 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     6121 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     6181 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     6241 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6301 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     6361 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     6421 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     6481 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6541 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6601 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6661 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     6721 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6781 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6841 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6901 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6961 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     7021 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     7081 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     7141 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     7201 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     7261 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     7321 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7381 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     7441 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7501 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7561 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7621 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7681 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7741 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7801 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7861 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7921 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7981 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     8041 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     8101 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     8161 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     8221 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
