LOCUS       sequence-356018-        8735 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 (includes a large 
            portion of the Slc8b1 3'UTR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8735)
  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 8735)
  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.3'UTR
FEATURES             Location/Qualifiers
     source          1..8735
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   4..133
                     /label=AAV2 ITR
     promoter        439..716
                     /label=chicken beta-actin promoter
     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    4562..5150
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4615..4635)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5033..5044)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5177..5194)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5183..5407
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(5617..5813)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(5625..5835)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5631..5766
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5931..5951
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(6192..6780)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6272..6291)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6951..7742,7743..7811))
                     /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     7574..7593
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7812..7916)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7984..8002
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8040..8062)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8162..8181
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8395..8414
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        8700..8718
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     8700..8717
                     /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 gggsgrggcg grgaggtgcg 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 agcttctgaa
     3781 gctgcttggc ctagaggtgt gggggcgatt ctgctagcct cctgaggggg aggtgtgggg
     3841 agggggaccc tctgtggtcc ccgtggatct cctgagaaga tagtcactgg cagagctctg
     3901 catgggtgag aaggtcctga ctgccggcac ctaacagcct tagtgtgggg atctggaggc
     3961 tggctttgct ggggacaatc ccgggtagga atgatgggat ctaaatgaca ccggaggctc
     4021 tggggggagg gcagcctttc agtcagcccc catgcctgct gggctctggg cagccctgca
     4081 gttccctctc tggctcttcc actctctgtg gggtcctgcg tacctacagg gtggcctgaa
     4141 aacagactcc acacgtggaa acaagactgg gtttctcagc ttccgtgtca agtcagccag
     4201 aggaaagagg tcgagttgac agcagcaggc actgcccttc ctagttggtg gctgccatgt
     4261 tggactgtgg atctaaacac ttctagagct ttgtggtcca aactctggct ctcccgtctg
     4321 caaaacagga gccagcctgg gctctgtgcc tacctcaagg ggagctgggg gttgggggga
     4381 ctgaccctcc ccagggaggt cttacaagta gtgtgaccag tcttttgttt gtttgtttgg
     4441 ttggttgttt tttggagaca gggtttctct gtgtagccct ggctgtcctg gaactcactc
     4501 tgtagaccag gctggccttg aactcagaaa tctgcctgcc tttgcctcca agcttatcga
     4561 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     4621 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     4681 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     4741 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     4801 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     4861 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     4921 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     4981 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     5041 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     5101 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     5161 tcgactcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     5221 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     5281 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     5341 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     5401 tctatggctt ctgaggcgga aagaaccagc tggggctcga ctagagcatg gctacgtaga
     5461 taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac
     5521 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     5581 gggcggcctc agtgagcgag cgagcgcgca gagctttttg caaaagccta ggcctccaaa
     5641 aaagcctcct cactacttct ggaatagctc agaggccgag gcggcctcgg cctctgcata
     5701 aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg
     5761 cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg
     5821 catacttctg cctgctgggg rgsswgkkrr skkyssrmmy mtggttgctg actaattgag
     5881 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg
     5941 acacacattc cacagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6001 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     6061 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     6121 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     6181 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     6241 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6301 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6361 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     6421 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     6481 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6541 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6601 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6661 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6721 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6781 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6841 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6901 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6961 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     7021 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     7081 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     7141 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     7201 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     7261 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     7321 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     7381 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     7441 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     7501 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7561 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7621 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7681 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7741 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7801 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7861 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7921 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     7981 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     8041 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     8101 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     8161 gcggcatcag agcagattgt actgagagtg caccattcga cgctctccct tatgcgactc
     8221 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     8281 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     8341 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     8401 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     8461 tccggcgtag aggatctggc gatgaccctg ctgattggtt cgctgaccat ttccgggtgc
     8521 gggacggcgt taccagaaac tcagaaggtt cgtccaacca aaccgactct gacggcagtt
     8581 tacgagagag atgatagggt ctgcttcagt aagccagatg ctacacaatt aggcttgtac
     8641 atattgtcgt tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga
     8701 tttaggtgac actatagaat acacggaatt aattc
//
