LOCUS       sequence-356032-        6475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Mcu-targeted shRNA under control of the U6 promoter and 
            hrGFP under control of the synthetic CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6475)
  AUTHORS   Qiu J, Tan YW, Hagenston AM, Martel MA, Kneisel N, Skehel PA, Wyllie
            DJ, Bading H, Hardingham GE
  TITLE     Mitochondrial calcium uniporter Mcu controls excitotoxicity and is 
            transcriptionally repressed by neuroprotective nuclear calcium 
            signals.
  JOURNAL   Nat Commun. 2013;4:2034. doi: 10.1038/ncomms3034.
  PUBMED    23774321
REFERENCE   2  (bases 1 to 6475)
  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: "Nat 
            Commun."; date: "2013"; volume: "4:2034. doi"; pages: " 
            10.1038/ncomms3034"
COMMENT     Sequence Label: pAAV-shMcu-1
FEATURES             Location/Qualifiers
     source          1..6475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   4..133
                     /label=AAV2 ITR
     promoter        298..546
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     298..318
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     469..488
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     1404..1426
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1488..1507
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1547..2266
                     /codon_start=1
                     /product="humanized Renilla green fluorescent protein"
                     /label=hrGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGN
                     QLVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYE
                     DGGLVEIRSDINLIEEMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVG
                     QVILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAI
                     AQLTSLGKPLGSLHEWV"
     misc_feature    2298..2886
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2351..2371)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2769..2780)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2913..2930)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2919..3143
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3353..3682)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3367..3502
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3667..3687
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3928..4516)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4008..4027)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4687..5478,5479..5547))
                     /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     5310..5329
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5548..5652)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5720..5738
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5776..5798)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5898..5917
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6131..6150
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6440..6458
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6440..6457
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 tagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
       61 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
      121 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
      181 tctaggtacc ctcgaggagg agaagcatga attccccagt ggaaagacgc gcaggcaaaa
      241 cgcaccacgt gacggagcgt gaccgcgcgc cgagcgcgcg ccaaggtcgg gcaggaagag
      301 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
      361 attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
      421 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
      481 cttaccgtaa cttgaaagta tttcgatttc ttgggtttat atatcttgtg gaaaggacgc
      541 gggatcccct agggaataaa gggatcttaa ttcaagagat taagatccct ttattcccta
      601 tttttggaaa agcttggcac tggccgtact agtggtacca ttgacgtcaa taatgacgta
      661 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      721 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      781 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      841 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      901 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      961 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggcgg ggcgaggcgg
     1021 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
     1081 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
     1141 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1201 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1261 cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc
     1321 cgggagggcc ctttgtgcgg ggggagcggc tcggggctgt ccgcgggggg acggctgcct
     1381 tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc
     1441 ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt
     1501 tattgtgctg tctcatcatt ttggcaaaga attggatcga tccaccatgg tgagcaagca
     1561 gatcctgaag aacaccggcc tgcaggagat catgagcttc aaggtgaacc tggagggcgt
     1621 ggtgaacaac cacgtgttca ccatggaggg ctgcggcaag ggcaacatcc tgttcggcaa
     1681 ccagctggtg cagatccgcg tgaccaaggg cgcccccctg cccttcgcct tcgacatcct
     1741 gagccccgcc ttccagtacg gcaaccgcac cttcaccaag taccccgagg acatcagcga
     1801 cttcttcatc cagagcttcc ccgccggctt cgtgtacgag cgcaccctgc gctacgagga
     1861 cggcggcctg gtggagatcc gcagcgacat caacctgatc gaggagatgt tcgtgtaccg
     1921 cgtggagtac aagggccgca acttccccaa cgacggcccc gtgatgaaga agaccatcac
     1981 cggcctgcag cccagcttcg aggtggtgta catgaacgac ggcgtgctgg tgggccaggt
     2041 gatcctggtg taccgcctga acagcggcaa gttctacagc tgccacatgc gcaccctgat
     2101 gaagagcaag ggcgtggtga aggacttccc cgagtaccac ttcatccagc accgcctgga
     2161 gaagacctac gtggaggacg gcggcttcgt ggagcagcac gagaccgcca tcgcccagct
     2221 gaccagcctg ggcaagcccc tgggcagcct gcacgagtgg gtgtaatagg aattcgatat
     2281 caagctagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     2341 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     2401 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     2461 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     2521 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     2581 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     2641 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     2701 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     2761 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     2821 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     2881 ccccgcatcg ataccgtcga ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     2941 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3001 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc gttctattct
     3061 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3121 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgactag
     3181 agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag
     3241 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     3301 aggtcgcccg acgcccgggc ggcctcagtg agcgagcgag cgcgcagagc tttttgcaaa
     3361 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gccgaggcgg
     3421 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
     3481 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat
     3541 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg
     3601 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     3661 ttccacaccc taactgacac acattccaca gctgcattaa tgaatcggcc aacgcgcggg
     3721 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3781 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3841 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3901 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3961 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4021 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4081 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4141 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4201 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4261 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4321 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4381 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4441 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4501 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4561 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4621 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4681 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4741 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4801 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4861 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4921 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4981 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5041 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5101 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5161 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5221 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5281 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5341 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5401 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5461 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5521 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5581 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5641 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     5701 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     5761 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     5821 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     5881 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc attcgacgct
     5941 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     6001 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     6061 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     6121 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     6181 tgccggccac gatgcgtccg gcgtagagga tctggctagc gatgaccctg ctgattggtt
     6241 cgctgaccat ttccgggtgc gggacggcgt taccagaaac tcagaaggtt cgtccaacca
     6301 aaccgactct gacggcagtt tacgagagag atgatagggt ctgcttcagt aagccagatg
     6361 ctacacaatt aggcttgtac atattgtcgt tagaacgcgg ctacaattaa tacataacct
     6421 tatgtatcat acacatacga tttaggtgac actatagaat acacggaatt aattc
//
