LOCUS       sequence-356033-        6511 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 6511)
  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 6511)
  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-2
FEATURES             Location/Qualifiers
     source          1..6511
                     /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     1440..1462
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1524..1543
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1583..2302
                     /codon_start=1
                     /product="humanized Renilla green fluorescent protein"
                     /label=hrGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGN
                     QLVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYE
                     DGGLVEIRSDINLIEEMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVG
                     QVILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAI
                     AQLTSLGKPLGSLHEWV"
     misc_feature    2334..2922
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2387..2407)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2805..2816)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2949..2966)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2955..3179
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3389..3718)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3403..3538
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3703..3723
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3964..4552)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4044..4063)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4723..5514,5515..5583))
                     /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     5346..5365
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5584..5688)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5756..5774
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5812..5834)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5934..5953
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6167..6186
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6476..6494
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6476..6493
                     /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 gggatccccg ggcttagcga gtcttgtctt caagagagac aagactcgct aagccctttt
      601 tggaaaagct tggcactggc cgtactagtg gtaccattga cgtcaataat gacgtatgtt
      661 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      721 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      781 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      841 acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg
      901 ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt
      961 ttttaattat tttgtgcagc gatgggggcg gggggggggg gggggcgcgc gccrggsggg
     1021 gsggggsggg gsgrggggsg gggsggggsg rggsggrgag gtgcggcggc agccaatcag
     1081 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
     1141 aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc
     1201 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
     1261 gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt
     1321 tcttttctgt ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg
     1381 agcggctcgg ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg
     1441 gttcggcttc tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt
     1501 cttctttttc ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg
     1561 caaagaattg gatcgatcca ccatggtgag caagcagatc ctgaagaaca ccggcctgca
     1621 ggagatcatg agcttcaagg tgaacctgga gggcgtggtg aacaaccacg tgttcaccat
     1681 ggagggctgc ggcaagggca acatcctgtt cggcaaccag ctggtgcaga tccgcgtgac
     1741 caagggcgcc cccctgccct tcgccttcga catcctgagc cccgccttcc agtacggcaa
     1801 ccgcaccttc accaagtacc ccgaggacat cagcgacttc ttcatccaga gcttccccgc
     1861 cggcttcgtg tacgagcgca ccctgcgcta cgaggacggc ggcctggtgg agatccgcag
     1921 cgacatcaac ctgatcgagg agatgttcgt gtaccgcgtg gagtacaagg gccgcaactt
     1981 ccccaacgac ggccccgtga tgaagaagac catcaccggc ctgcagccca gcttcgaggt
     2041 ggtgtacatg aacgacggcg tgctggtggg ccaggtgatc ctggtgtacc gcctgaacag
     2101 cggcaagttc tacagctgcc acatgcgcac cctgatgaag agcaagggcg tggtgaagga
     2161 cttccccgag taccacttca tccagcaccg cctggagaag acctacgtgg aggacggcgg
     2221 cttcgtggag cagcacgaga ccgccatcgc ccagctgacc agcctgggca agcccctggg
     2281 cagcctgcac gagtgggtgt aataggaatt cgatatcaag ctagcttatc gataatcaac
     2341 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     2401 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     2461 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     2521 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     2581 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     2641 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     2701 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg
     2761 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     2821 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     2881 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgtcgactcg
     2941 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3001 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3061 tgcatcgcat tgtctgagta ggtgtcgttc tattctgggg ggtggggtgg ggcaggacag
     3121 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3181 ttctgaggcg gaaagaacca gctggggctc gactagagca tggctacgta gataagtagc
     3241 atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc
     3301 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggcggcc
     3361 tcagtgagcg agcgagcgcg cagagctttt tgcaaaagcc taggcctcca aaaaagcctc
     3421 ctcactactt ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa
     3481 aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg
     3541 gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc
     3601 tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc
     3661 tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat
     3721 tccacagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3781 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3841 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3901 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3961 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4021 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4081 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4141 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4201 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4261 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4321 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4381 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4441 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4501 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4561 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4621 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4681 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4741 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4801 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4861 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4921 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4981 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5041 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5101 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5161 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5221 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5281 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5341 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5401 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5461 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5521 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5581 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5641 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5701 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     5761 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     5821 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     5881 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     5941 agagcagatt gtactgagag tgcaccattc gacgctctcc cttatgcgac tcctgcatta
     6001 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     6061 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     6121 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     6181 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     6241 agaggatctg gctagcgatg accctgctga ttggttcgct gaccatttcc gggtgcggga
     6301 cggcgttacc agaaactcag aaggttcgtc caaccaaacc gactctgacg gcagtttacg
     6361 agagagatga tagggtctgc ttcagtaagc cagatgctac acaattaggc ttgtacatat
     6421 tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac atacgattta
     6481 ggtgacacta tagaatacac ggaattaatt c
//
