LOCUS       sequence-444154-        5599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone under tet-operator for RNA interference, with YFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5599)
  AUTHORS   Kim J, de Haro M, Al-Ramahi I, Garaicoechea LL, Jeong HH, Sonn JY, 
            Tadros B, Liu Z, Botas J, Zoghbi HY
  TITLE     Evolutionarily conserved regulators of tau identify targets for new 
            therapies.
  JOURNAL   Neuron. 2023 Mar 15;111(6):824-838.e7. doi: 
            10.1016/j.neuron.2022.12.012. Epub 2023 Jan 6.
  PUBMED    36610398
REFERENCE   2  (bases 1 to 5599)
  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: 
            "10.1016/j.neuron.2022.12.012"; journalName: "Neuron"; date: 
            "2023-03-15- 15"; volume: "111"; issue: "6"; pages: "824-838.e7"
COMMENT     Sequence Label: Luciferase shRNA-TRE
FEATURES             Location/Qualifiers
     source          1..5599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1204..1222
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1260..1282)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1382..1401
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1615..1634
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1924..1942
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1924..1941
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     repeat_region   1963..2092
                     /label=AAV2 ITR
     promoter        2149..2526
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    2157..2175
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2193..2211
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2229..2247
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2265..2283
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2301..2319
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2337..2355
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2373..2391
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2436..2460
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2533..2542
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2539..2541,2542..2544,2545..3258)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2584..2605)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2845..2864)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3192..3213
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           3444..3458
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           3780..3794
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     complement(3996..4013)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4002..4226
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(4436..4765)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4450..4585
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4750..4770
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5011..5599)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5091..5110)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     1201 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     1261 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     1321 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     1381 gcggcatcag agcagattgt actgagagtg caccattcga cgctctccct tatgcgactc
     1441 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     1501 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     1561 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     1621 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     1681 tccggcgtag aggatctggc tagcgatgac cctgctgatt ggttcgctga ccatttccgg
     1741 gtgcgggacg gcgttaccag aaactcagaa ggttcgtcca accaaaccga ctctgacggc
     1801 agtttacgag agagatgata gggtctgctt cagtaagcca gatgctacac aattaggctt
     1861 gtacatattg tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat
     1921 acgatttagg tgacactata gaatacacgg aattaattct agctgcgcgc tcgctcgctc
     1981 actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
     2041 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc cttgtagtta
     2101 atgattaacc cgccatgcta cttatctacg tagccatgct ctagcgtcga gtttactccc
     2161 tatcagtgat agagaacgta tgaagagttt actccctatc agtgatagag aacgtatgca
     2221 gactttactc cctatcagtg atagagaacg tataaggagt ttactcccta tcagtgatag
     2281 agaacgtatg accagtttac tccctatcag tgatagagaa cgtatctaca gtttactccc
     2341 tatcagtgat agagaacgta tatccagttt actccctatc agtgatagag aacgtataag
     2401 ctttaggcgt gtacggtggg cgcctataaa agcagagctc gtttagtgaa ccgtcagatc
     2461 gcctggagca attccacaac acttttgtct tataccaact ttccgtacca cttcctaccc
     2521 tcgtaaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     2581 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     2641 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     2701 cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg cttcgcccgc
     2761 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2821 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     2881 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2941 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     3001 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     3061 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     3121 ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga ccccaacgag
     3181 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     3241 gacgagctgt acaagtaaag cggccgcctc gactagggat aacagggtaa ttgtttgaat
     3301 gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta
     3361 cttcgacttc ttaacccaac agaaggctcg agaaggtata ttgctgttga cagtgagcgc
     3421 ccgcctgaag tctctgatta atagtgaagc cacagatgta ttaatcagag acttcaggcg
     3481 gttgcctact gcctcggact tcaaggggct agatatcgag caattatctt gtttactaaa
     3541 actgaatacc ttgctatctc tttgatacat ttttacaaag ctgaattaaa atggtataaa
     3601 ttaaatcact ttgtgctcat cacgaactgt ttgaatgagg cttcagtact ttacagaatc
     3661 gttgcctgca catcttggaa acacttgctg ggattacttc gacttcttaa cccaacagaa
     3721 gggaattcaa ggtatattgc tgttgacagt gagcgcccgc ctgaagtctc tgattaatag
     3781 tgaagccaca gatgtattaa tcagagactt caggcggttg cctactgcct cggacttcaa
     3841 ggggctagaa ttcgagcaat tatcttgttt actaaaactg aataccttgc tatctctttg
     3901 atacattttt acaaagctga attaaaatgg tataaattaa atcacttttt tcaattgacg
     3961 cgtaagctta tcgataccgt cgactcgctg atcagcctcg actgtgcctt ctagttgcca
     4021 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     4081 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     4141 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     4201 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctcgac
     4261 tagagcatgg ctacgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc
     4321 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     4381 caaaggtcgc ccgacgcccg ggcggcctca gtgagcgagc gagcgcgcag agctttttgc
     4441 aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg
     4501 cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg
     4561 aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact
     4621 aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc
     4681 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
     4741 actttccaca ccctaactga cacacattcc acagctgcat taatgaatcg gccaacgcgc
     4801 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4861 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4921 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4981 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5041 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5101 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5161 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5221 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5281 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5341 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5401 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5461 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5521 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5581 cagaaaaaaa ggatctcaa
//
