LOCUS       sequence-350456-        6434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Producing AAV that encodes mouse Rps6ka2 (Rsk3) shRNA-2 with miR-E 
            backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6434)
  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 6434)
  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_shRps6ka2-2
FEATURES             Location/Qualifiers
     source          1..6434
                     /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"
     repeat_region   1310..1439
                     /label=AAV2 ITR
     promoter        complement(1445..1774)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1459..1594
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1759..1779
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2020..2608)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2100..2119)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2779..3570,3571..3639))
                     /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     3402..3421
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3640..3744)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3812..3830
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3868..3890)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3990..4009
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4223..4242
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4532..4550
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4532..4549
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     repeat_region   4571..4700
                     /label=AAV2 ITR
     promoter        5006..5283
                     /label=chicken beta-actin promoter
     primer_bind     5563..5585
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     5647..5666
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      5706..5715
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5712..5714,5715..5717,5718..6431)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5757..5778)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6018..6037)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6365..6386
                     /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 agtgagcgat gaaaccaagt aacattctga
      181 tagtgaagcc acagatgtat cagaatgtta cttggtttca gtgcctactg 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 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcaga
     1441 gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg aatagctcag
     1501 aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca tggggcggag
     1561 aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc gggactatgg
     1621 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     1681 ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg
     1741 agcctgggga ctttccacac cctaactgac acacattcca cagctgcatt aatgaatcgg
     1801 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     1861 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     1921 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     1981 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2041 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2101 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2161 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2221 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2281 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2341 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2401 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     2461 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     2521 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     2581 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     2641 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     2701 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     2761 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     2821 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     2881 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     2941 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3001 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3061 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3121 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3181 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3241 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3301 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3361 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     3421 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     3481 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     3541 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     3601 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     3661 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     3721 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     3781 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     3841 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     3901 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     3961 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     4021 ccattcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     4081 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     4141 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     4201 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     4261 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatctggcta gcgatgaccc
     4321 tgctgattgg ttcgctgacc atttccgggt gcgggacggc gttaccagaa actcagaagg
     4381 ttcgtccaac caaaccgact ctgacggcag tttacgagag agatgatagg gtctgcttca
     4441 gtaagccaga tgctacacaa ttaggcttgt acatattgtc gttagaacgc ggctacaatt
     4501 aatacataac cttatgtatc atacacatac gatttaggtg acactataga atacacggaa
     4561 ttaattctag ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg
     4621 ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa
     4681 ctccatcact aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta
     4741 gccatgctct aggtaccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     4801 gggactttcc attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta
     4861 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     4921 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     4981 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
     5041 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
     5101 gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg
     5161 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
     5221 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
     5281 gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
     5341 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
     5401 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
     5461 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggctgtc
     5521 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     5581 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     5641 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttggatccac
     5701 cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     5761 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     5821 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     5881 cctggcccac cctcgtgacc accttcggct acggcctgca gtgcttcgcc cgctaccccg
     5941 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     6001 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     6061 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     6121 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     6181 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     6241 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     6301 ccgacaacca ctacctgagc taccagtccg ccctgagcaa agaccccaac gagaagcgcg
     6361 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     6421 tgtacaagta aagc
//
