LOCUS       sequence-354144-        6422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Producing AAV that encodes mouse Rps6ka2 (Rsk3) shRNA-3 with miR-E 
            backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6422)
  AUTHORS   Lee WS, Lavery L, Rousseaux MWC, Rutledge EB, Jang Y, Wan YW, Wu SR,
            Kim W, Al-Ramahi I, Rath S, Adamski CJ, Bondar VV, Tewari A, 
            Soleimani S, Mota S, Yalamanchili HK, Orr HT, Liu Z, Botas J, Zoghbi
            HY
  TITLE     Dual targeting of brain region-specific kinases potentiates 
            neurological rescue in Spinocerebellar ataxia type 1.
  JOURNAL   EMBO J. 2021 Apr 1;40(7):e106106. doi: 10.15252/embj.2020106106. 
            Epub 2021 Mar 11.
  PUBMED    33709453
REFERENCE   2  (bases 1 to 6422)
  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"; journalName: "EMBO J."; 
            date: "2021-04-1"; volume: "40(7):e106106. doi"; pages: " 
            10.15252/embj.2020106106. Epub 2021 Mar 11"
COMMENT     Sequence Label: AAV.CBA.YFP.miR-E_shRps6ka2-3
FEATURES             Location/Qualifiers
     source          1..6422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     ncRNA           183..197
                     /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"
     misc_feature    379..967
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(432..452)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(850..861)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(994..1011)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1000..1224
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(1434..1763)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1448..1583
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1748..1768
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2009..2597)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2089..2108)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2768..3559,3560..3628))
                     /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     3391..3410
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3629..3733)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3801..3819
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3857..3879)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3979..3998
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4212..4231
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4521..4539
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4521..4538
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     repeat_region   4560..4689
                     /label=AAV2 ITR
     promoter        4995..5272
                     /label=chicken beta-actin promoter
     primer_bind     5551..5573
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     5635..5654
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      5694..5703
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5700..5702,5703..5705,5706..6419)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5745..5766)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6006..6025)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6353..6374
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ggccgcctcg actagggata acagggtaat tgtttgaatg aggcttcagt actttacaga
       61 atcgttgcct gcacatcttg gaaacacttg ctgggattac ttcgacttct taacccaaca
      121 gaaggctcga gaaggtatat tgctgttgac agtgagcgcc acccgttctt tgtcaccata
      181 tagtgaagcc acagatgtat atggtgacaa agaacgggtg atgcctactg cctcggactt
      241 caaggggcta gaattcgagc aattatcttg tttactaaaa ctgaatacct tgctatctct
      301 ttgatacatt tttacaaagc tgaattaaaa tggtataaat taaatcactt ttttcaattg
      361 acgcgtaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
      421 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
      481 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
      541 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
      601 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
      661 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
      721 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
      781 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
      841 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
      901 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
      961 tccccgcatc gataccgtcg actcgctgat cagcctcgac tgtgccttct agttgccagc
     1021 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     1081 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     1141 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     1201 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgacta
     1261 gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta
     1321 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     1381 aaggtcgccc gacgcccggg cggcctcagt gagcgagcga gcgcgcagag ctttttgcaa
     1441 aagcctaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga ggccgaggcg
     1501 gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa
     1561 ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa
     1621 ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg
     1681 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
     1741 tttccacacc ctaactgaca cacattccac agctgcatta atgaatcggc caacgcgcgg
     1801 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     1861 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1921 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     1981 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2041 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2101 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2161 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2221 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2281 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2341 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2401 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2461 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2521 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2581 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2641 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2701 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2761 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2821 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2881 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     2941 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3001 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3061 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3121 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3181 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3241 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3301 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3361 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3421 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3481 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3541 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3601 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3661 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3721 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     3781 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     3841 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     3901 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     3961 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac cattcgacgc
     4021 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     4081 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     4141 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     4201 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     4261 atgccggcca cgatgcgtcc ggcgtagagg atctggctag cgatgaccct gctgattggt
     4321 tcgctgacca tttccgggtg cgggacggcg ttaccagaaa ctcagaaggt tcgtccaacc
     4381 aaaccgactc tgacggcagt ttacgagaga gatgataggg tctgcttcag taagccagat
     4441 gctacacaat taggcttgta catattgtcg ttagaacgcg gctacaatta atacataacc
     4501 ttatgtatca tacacatacg atttaggtga cactatagaa tacacggaat taattctagc
     4561 tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg
     4621 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
     4681 ggggttcctt gtagttaatg attaacccgc catgctactt atctacgtag ccatgctcta
     4741 ggtaccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     4801 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     4861 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     4921 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     4981 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
     5041 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
     5101 gggggggggg ggggggcgcg cgccrggsgg ggsggggsgg ggsgrggggs ggggsggggs
     5161 grggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
     5221 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
     5281 tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
     5341 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     5401 taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg aaagccttga
     5461 ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggctgtccg cggggggacg
     5521 gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct
     5581 ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg
     5641 tgctggttat tgtgctgtct catcattttg gcaaagaatt ggatccaccg gtcgccacca
     5701 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     5761 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     5821 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     5881 tcgtgaccac cttcggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc
     5941 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     6001 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     6061 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     6121 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     6181 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     6241 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     6301 acctgagcta ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     6361 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa
     6421 gc
//
