LOCUS       sequence-355961-        6827 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NCLX-targeted shRNA under control of the U6 promoter and 
            mCherry under control of the CaMKIIa promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6827)
  AUTHORS   Hagenston AM, Yan J, Bas-Orth C, Tan Y, Sekler I, Bading H
  TITLE     Disrupted expression of mitochondrial NCLX sensitizes neuroglial 
            networks to excitotoxic stimuli and renders synaptic activity toxic.
  JOURNAL   J Biol Chem. 2021 Dec 20:101508. doi: 10.1016/j.jbc.2021.101508.
  PUBMED    34942149
REFERENCE   2  (bases 1 to 6827)
  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: "J Biol 
            Chem. 2021 Dec 20:101508. doi: 10.1016/j.jbc.2021.101508."
COMMENT     Sequence Label: pAAV-shNCLX-2
FEATURES             Location/Qualifiers
     source          1..6827
                     /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"
     CDS             1892..2602
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2037..2055)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2305..2324
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2650..3238
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2703..2723)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3121..3132)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3265..3282)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3271..3495
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3705..4034)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3719..3854
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4019..4039
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(4280..4868)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4360..4379)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5039..5830,5831..5899))
                     /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     5662..5681
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5900..6004)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6072..6090
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6128..6150)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6250..6269
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6483..6502
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6792..6810
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6792..6809
                     /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 gggatccccc gacaaggacg atcggaattg ttcaagagac aattccgatc gtccttgtcg
      601 tttttggaaa agcttggcac tggccgtact agaaaatgag atgggggtgc agagagcttc
      661 ctcagtgacc tgcccagggt cacatcagaa atgtcagagc tagaacttga actcagatta
      721 ctaatcttaa attccatgcc ttgggggcat gcaagtacga tatacagaag gagtgaactc
      781 attagggcag atgaccaatg agtttaggaa agaagagtcc agggcagggt acatctacac
      841 cacccgccca gccctgggtg agtccagcca cgttcacctc attatagttg cctctctcca
      901 gtcctacctt gacgggaagc acaagcagaa actgggacag gagccccagg agaccaaatc
      961 ttcatggtcc ctctgggagg atgggtgggg agagctgtgg cagaggcctc aggaggggcc
     1021 ctgctgctca gtggtgacag ataggggtga gaaagcagac agagtcattc cgtcagcatt
     1081 ctgggtctgt ttggtacttc ttctcacgct aaggtggcgg tgtgatatgc acaatggcta
     1141 aaaagcaggg agagctggaa agaaacaagg acagagacag aggccaagtc aaccagacca
     1201 attcccagag gaagcaaaga aaccattaca gagactacaa gggggaaggg aaggagagat
     1261 gaattagctt cccctgtaaa ccttagaacc cagctgttgc cagggcaacg gggcaatacc
     1321 tgtctcttca gaggagatga agttgccagg gtaactacat cctgtctttc tcaaggacca
     1381 tcccagaatg tggcacccac tagccgttac catagcaact gcctctttgc cccacttaat
     1441 cccatcccgt ctgttaaaag ggccctatag ttggaggtgg gggaggtagg aagagcgatg
     1501 atcacttgtg gactaagttt gttcgcatcc ccttctccaa ccccctcagt acatcaccct
     1561 gggggaacag ggtccacttg ctcctgggcc cacacagtcc tgcagtattg tgtatataag
     1621 gccagggcaa agaggagcag gttttaaagt gaaaggcagg caggtgttgg ggaggcagtt
     1681 accggggcaa cgggaacagg gcgtttcgga ggtggttgcc atggggacct ggatgctgac
     1741 gaaggctcgc gaggctgtga gcagccacag tgccctgctc agaagcccca agctcgtcag
     1801 tcaagccggt tctccgtttg cactcaggag cacgggcagg cgagtggccc ctagttctgg
     1861 gggcagcggg ggatcaggtg gcagcggtgg catggtgagc aagggcgagg aggataacat
     1921 ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     1981 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     2041 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     2101 catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     2161 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     2221 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     2281 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     2341 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
     2401 gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
     2461 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa
     2521 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     2581 catggacgag ctgtacaagt agggtggcag cggtggcact cgagtaagct atcaagctag
     2641 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     2701 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     2761 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     2821 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     2881 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     2941 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     3001 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     3061 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     3121 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     3181 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     3241 cgataccgtc gactcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     3301 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     3361 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3421 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     3481 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgact agagcatggc
     3541 tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag
     3601 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     3661 cgacgcccgg gcggcctcag tgagcgagcg agcgcgcaga gctttttgca aaagcctagg
     3721 cctccaaaaa agcctcctca ctacttctgg aatagctcag aggccgaggc ggcctcggcc
     3781 tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga
     3841 gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc
     3901 atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac
     3961 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     4021 cctaactgac acacattcca cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4081 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4141 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4201 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4261 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4321 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4381 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4441 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4501 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4561 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4621 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4681 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4741 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4801 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4861 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4921 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4981 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5041 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5101 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5161 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5221 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5281 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5341 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5401 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5461 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5521 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5581 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5641 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5701 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5761 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5821 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5881 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5941 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6001 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6061 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     6121 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     6181 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     6241 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattcgacg ctctccctta
     6301 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     6361 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     6421 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     6481 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     6541 acgatgcgtc cggcgtagag gatctggcta gcgatgaccc tgctgattgg ttcgctgacc
     6601 atttccgggt gcgggacggc gttaccagaa actcagaagg ttcgtccaac caaaccgact
     6661 ctgacggcag tttacgagag agatgatagg gtctgcttca gtaagccaga tgctacacaa
     6721 ttaggcttgt acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc
     6781 atacacatac gatttaggtg acactataga atacacggaa ttaattc
//
