LOCUS       sequence-355952-        7955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP and murine NCLX (separated by a T2A cleavage site) 
            under control of the synthetic CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7955)
  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 7955)
  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-EGFP.T2A.NCLX
FEATURES             Location/Qualifiers
     source          1..7955
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   4..133
                     /label=AAV2 ITR
     primer_bind     995..1017
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1079..1098
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(1151..1153,1154..1156,1157..1867)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1151..1153,1154..1156,1157..1867)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1196..1217)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1457..1476)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1804..1825
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1874..1900
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1907..1960
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3731..3760
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    3782..4370
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3835..3855)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4253..4264)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4397..4414)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4403..4627
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(4837..5033)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(4845..5055)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4851..4986
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5151..5171
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5412..6000)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5492..5511)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6171..6962,6963..7031))
                     /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     6794..6813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7032..7136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7204..7222
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7260..7282)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7382..7401
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7615..7634
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7920..7938
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     7920..7937
                     /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 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      241 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      301 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      361 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      421 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
      481 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
      541 gggcgggggg gggggggggg cgcgcgccrg gsggggsggg gsggggsgrg gggsggggsg
      601 gggsgrggsg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
      661 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
      721 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
      781 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
      841 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc
      901 ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg
      961 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     1021 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     1081 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatct aagctagcga
     1141 attcgtcacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1201 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1261 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1321 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1381 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1441 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1501 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1561 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     1621 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     1681 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1741 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     1801 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     1861 gtacaaggct agctacccat acgatgtccc agattacgct ggatccgagg gcagaggaag
     1921 tcttctaaca tgcggtgacg tggaggagaa tcccggccct accgggacca tggcaagcag
     1981 atggctggct ctgctctggg ctcctgtctt cctctgtgtg gctctgatac tggagacggc
     2041 gtctgggacc ggagacccat ccacaaaagc ccatggacac atccagtttt cagctggaag
     2101 tgtcaaccag actgccatgg cggattgccg agccgtgtgt ggcctgaaca catctgatcg
     2161 ctgtgacttt gtcaggagga atccggactg ccgcagcgag gcgggctacc tggactacct
     2221 tgagggcatc ttctgctact tcccccccaa cctcctccct ctggccatca ccctctacgt
     2281 tttctggctg ctttacctct ttctgatcct gggagtcacc gcggccaagt tcttctgccc
     2341 taacctgtca gccatctcca ctaacctcaa actctcccac aacgtggcag gtgtcacctt
     2401 cctggccttt ggaaatggcg ctccagacat cttcagtgct ttagtggctt tctcagaccc
     2461 acgtactgcc ggcctggcca tcggggctct gtttggtgca ggggtgctgg tcaccactgt
     2521 ggtggctgga ggcatcacca tcctgcatcc cttcatggct gcctccaggc ccttcctcag
     2581 ggacatcgct ttctacatgg tggctgtgtt cctaaccttc actgcactct atctcggcag
     2641 gatcacgctg acgtgggcgc tgggttacct gggcctctac gtgttctacg tggtcacggt
     2701 catcatctgc acttgggtct accaacggca gcgaagcagg tctctggtcc actccatatc
     2761 ggagacacca gagttgctgt ctgagtcaga ggaggaccag atgtcttcca acaccaacag
     2821 ctatgactat ggagatgaat accggcctct gttgctgggt cgggagacca ctgtccagat
     2881 cctgatccaa gccctgaatc ccttggacta caggaagtgg agaactcagt cgatatcctg
     2941 gagggtcctg aaggtagtca agttgcctgt ggagttcttg ttgctgctca cagtaccggt
     3001 tgtggacccc gacaaggacg atcggaattg gaaacggcca ctcaactgtc tgcagctggt
     3061 catcagcccc ctggtcctgg tcctgaccct gcagtcgggg gtctatggca tctatgagat
     3121 cggcggtctc cttcctgtct gggctgtggt ggtgatcgtg ggcacagcgc tggcttcagt
     3181 gaccttcttt gccacgtcta acagagaacc ccctagactg cactggctct ttgctttcct
     3241 gggtttcctg accagtgccc tgtggatcaa tgcagccgcc acagaggtgg tgaacatatt
     3301 acggtccctg ggtgtgatct tccgcctgag caacaccgtc ctagggctga ccctcctggc
     3361 ctggggaaac agcattggag atgccttctc agatttcacg ctggcccgcc aaggataccc
     3421 tcggatggcc ttctccgcct gtttcggggg catcatcttc aacatcctgg ttggtgtggg
     3481 gctgggctgc ttgctgcaga tcatcaggaa ccacgttgtg gaggtgaagc tggagccaga
     3541 cggattactg gtgtgggtgc tggccagtgc cctgggcctc agcttgatct tctccctggt
     3601 ctccgtgccg cttcagtgtt tccagctcag caaggcttac ggcctctgcc tcctcctctt
     3661 ctacatctgt ttccttgttg tggtcctgct cacagagttt ggggtgattc acctgaagaa
     3721 ggcggctctc gagcagaaac tcatctcaga agaggatctg gcagcataaa agcttatcga
     3781 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     3841 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     3901 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     3961 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     4021 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     4081 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     4141 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     4201 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     4261 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     4321 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     4381 tcgactcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     4441 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     4501 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     4561 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     4621 tctatggctt ctgaggcgga aagaaccagc tggggctcga ctagagcatg gctacgtaga
     4681 taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac
     4741 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4801 gggcggcctc agtgagcgag cgagcgcgca gagctttttg caaaagccta ggcctccaaa
     4861 aaagcctcct cactacttct ggaatagctc agaggccgag gcggcctcgg cctctgcata
     4921 aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg
     4981 cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg
     5041 catacttctg cctgctgggg rgsswgkkrr skkyssrmmy mtggttgctg actaattgag
     5101 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg
     5161 acacacattc cacagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5221 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5281 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5341 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5401 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5461 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5521 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5581 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5641 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5701 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5761 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5821 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5881 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5941 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6001 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6061 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7141 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     7201 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     7261 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     7321 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     7381 gcggcatcag agcagattgt actgagagtg caccattcga cgctctccct tatgcgactc
     7441 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     7501 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     7561 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     7621 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     7681 tccggcgtag aggatctggc gatgaccctg ctgattggtt cgctgaccat ttccgggtgc
     7741 gggacggcgt taccagaaac tcagaaggtt cgtccaacca aaccgactct gacggcagtt
     7801 tacgagagag atgatagggt ctgcttcagt aagccagatg ctacacaatt aggcttgtac
     7861 atattgtcgt tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga
     7921 tttaggtgac actatagaat acacggaatt aattc
//
