LOCUS       sequence-446526-        6081 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for Doxycycline inducible expression of 
            TRIM21 RING with a self-cleaving T2A-mCherry fluorescent expression 
            reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6081)
  AUTHORS   Benn J, Cheng S, Keeling S, Smith AE, Vaysburd MJ, Boken D, Miller 
            LVC, Katsinelos T, Franco C, Dupre E, Danis C, Landrieu I, Buee L, 
            Klenerman D, James LC, McEwan WA
  TITLE     Aggregate-selective removal of pathological tau by 
            clustering-activated degraders.
  JOURNAL   Science. 2024 Aug 30;385(6712):1009-1016. doi: 
            10.1126/science.adp5186. Epub 2024 Aug 29.
  PUBMED    39208111
REFERENCE   2  (bases 1 to 6081)
  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: 
            "10.1126/science.adp5186"; journalName: "Science"; date: 
            "2024-08-30- 30"; volume: "385"; issue: "6712"; pages: "1009-1016"
COMMENT     Sequence Label: pcDNA4TO T21R-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..6081
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     576..595
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(814..918)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(993..1012)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1184..1563
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1564..1767
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1718..1738
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    1769..1787
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1790..1808
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1818..1842
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1951..1960
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2242..2295
                     /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             2296..3006
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2441..2459)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2709..2728
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3038..3055)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3044..3268
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3314..3742
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3401..3420)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3611..3632
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3751..3771)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3756..4085
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3936..4071
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3998..4017
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4133..4180
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4199..4573
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    4703..4824
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4740..4759)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4794..4813
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4873..4889)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4873..4889)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4886..4908)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4897..4913
                     /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(4921..4927,4928..4945,4946..4951))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4966..4987
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5104..5121)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5275..5863)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5355..5374)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6034..6081,1..744,745..813))
                     /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"
ORIGIN
        1 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
       61 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
      121 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
      181 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
      241 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
      301 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
      361 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
      421 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
      481 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
      541 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
      601 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
      661 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
      721 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
      781 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
      841 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
      901 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
      961 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     1021 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     1081 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     1141 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
     1201 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     1261 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     1321 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     1381 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     1441 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     1501 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     1561 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
     1621 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
     1681 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
     1741 cggtgggagg tctatataag cagagctctc cctatcagtg atagagatct ccctatcagt
     1801 gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca
     1861 cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggac tctagcgttt
     1921 aaacttaagc ttggtaccga gctcggatcc gccaccatgg cttcagcagc acgcttgaca
     1981 atgatgtggg aggaggtcac atgccctatc tgcctggacc ccttcgtgga gcctgtgagc
     2041 atcgagtgtg gccacagctt ctgccaggaa tgcatctctc aggttgggaa aggtgggggc
     2101 agcgtctgtc ctgtgtgccg gcagcgcttt ctgctcaaga atctccggcc caatcgacag
     2161 ctagccaaca tggtgaacaa ccttaaagaa atcagccagg aggccagaga gggtggaggc
     2221 ggttcagaat tcggctccgg cgagggcagg ggaagtcttc taacatgcgg ggacgtggag
     2281 gaaaatcccg gcccaatggt gagcaagggc gaggaggata acatggccat catcaaggag
     2341 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag
     2401 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
     2461 ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag
     2521 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc
     2581 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
     2641 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
     2701 tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg
     2761 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc
     2821 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
     2881 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc
     2941 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     3001 aagtgatcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     3061 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3121 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3181 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3241 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3301 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3361 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3421 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3481 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3541 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3601 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3661 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3721 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3781 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3841 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3901 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3961 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     4021 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4081 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa
     4141 ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat
     4201 ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga
     4261 gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt
     4321 ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa
     4381 caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt
     4441 cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc
     4501 gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga
     4561 ggagcaggac tgacacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt
     4621 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     4681 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     4741 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4801 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     4861 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4921 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4981 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5041 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5101 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5161 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5221 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5281 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5341 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5401 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5461 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5521 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5581 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5641 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5701 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5761 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5821 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5881 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5941 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6001 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6061 tatctcagcg atctgtctat t
//
