LOCUS       sequence-450754-        8268 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses tau propagation ORF with V5 tag on S199D mutant 2N4R tau.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8268)
  TITLE     Hyman lab Tau2N4R plasmids
REFERENCE   2  (bases 1 to 8268)
  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-S199D.Tau2N4R-WPRE
FEATURES             Location/Qualifiers
     source          1..8268
                     /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(3859..3876)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3865..4089
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4135..4563
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4222..4241)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4432..4453
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4572..4592)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4577..4906
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4757..4892
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4819..4838
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4973..5767
                     /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(5027..5046)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5637..5656
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5941..6062
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5978..5997)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6032..6051
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6111..6127)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6111..6127)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6124..6146)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6135..6151
                     /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(6159..6165,6166..6183,6184..6189))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6204..6225
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6342..6359)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6513..7101)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6593..6612)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7272..8063,8064..8132))
                     /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     7895..7914
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8133..8237)
                     /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 ggctacagcg atcccggctc 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 cacgtctcca 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 cgatatctgc agaattccac cacactggac tagtggatcc gagctcggta ccaagcttaa
     3841 gtttaaaccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     3901 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     3961 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     4021 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     4081 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     4141 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4201 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4261 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4321 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     4381 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4441 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     4501 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4561 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     4621 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     4681 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     4741 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4801 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     4861 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     4921 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     4981 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     5041 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     5101 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     5161 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     5221 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     5281 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     5341 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     5401 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     5461 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     5521 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     5581 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     5641 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     5701 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     5761 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     5821 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     5881 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     5941 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     6001 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     6061 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     6121 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     6181 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     6241 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6301 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     6361 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6421 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6481 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     6541 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     6601 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     6661 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     6721 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6781 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6841 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6901 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6961 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     7021 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     7081 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     7141 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     7201 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     7261 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     7321 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     7381 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     7441 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     7501 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     7561 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     7621 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     7681 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     7741 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7801 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7861 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7921 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7981 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     8041 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     8101 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     8161 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     8221 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
