LOCUS       sequence-488279-        5919 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Mito-Timer fluorescent reporter, with miRNA-124 target sequence to 
            repress transgene expression in neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5919)
  AUTHORS   Deglon Lab
  TITLE     Deglon Lab Plasmids 2025
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5919)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-G1B3-Mito-Timer-WPRE-miR124T-SYNpolyA
FEATURES             Location/Qualifiers
     source          1..5919
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    819..843
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    2067..2091
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2124..2198
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      2226..2235
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2232..2909
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(2412..2432)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2824..2847
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(2932..2956)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2979..2995)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2980..2996)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     misc_feature    3009..3597
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3062..3082)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3480..3491)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3776..3824
                     /note="synthetic polyadenylation signal"
     repeat_region   3832..3972
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3832..3961
                     /label=AAV2 ITR
     rep_origin      4047..4502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4134..4153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4344..4365
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4519..4538)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4638..4660
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4698..4716)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4784..4888
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4889..4957,4958..5749)
                     /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"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggcctcta gtggatcccc cgggctgcag gaattatcac aagtttgtac aaaaaagcag
      841 gctccaccat gggaaccaat tcagtcgact ggatccggta ccgaattcga gctcggtacc
      901 agatctaaca tatcctggtg tggagtaggg gacgctgctc tgacagaggc tcgggggcct
      961 gagctggctc tgtgagctgg ggaggaggca gacagccagg ccttgtctgc aagcagacct
     1021 ggcagcattg ggctggccgc cccccagggc ctcctcttca tgcccagtga atgactcacc
     1081 ttggcacaga cacaatgttc ggggtgggca cagtgcctgc ttcccgccgc accccagccc
     1141 ccctcaaatg ccttccgaga agcccattga gcagggggct tgcattgcac cccagcctga
     1201 cagcctggca tcttgggata aaagcagcac agccccctag gggctgccct tgctgtgtgg
     1261 cgccaccggc ggtggagaac aaggctctat tcagcctgtg cccaggaaag gggatcaggg
     1321 gatgcccagg catggacagt gggtggcagg gggggagagg agggctgtct gcttcccaga
     1381 agtccaagga cacaaatggg tgaggggaga gctctcccca tagctgggct gcggcccaac
     1441 cccaccccct caggctatgc cagggggtgt tgccaggggc acccgatctt cctcttcatg
     1501 cccagtgaat gactcacctt ggcacagaca caatgttcgg ggtgggcaca gtgcctgctt
     1561 cccgccgcac cccagccccc ctcaaatgcc ttccgagaag cccattgagc aggcccgatc
     1621 ttcctcttca tgcccagtga atgactcacc ttggcacaga cacaatgttc ggggtgggca
     1681 cagtgcctgc ttcccgccgc accccagccc ccctcaaatg ccttccgaga agcccattga
     1741 gcaggcccga tcttcctctt catgcccagt gaatgactca ccttggcaca gacacaatgt
     1801 tcggggtggg cacagtgcct gcttcccgcc gcaccccagc ccccctcaaa tgccttccga
     1861 gaagcccatt gagcaggccc gggcatcgcc agtctagccc actccttcat aaagccctcg
     1921 catcccagga gcgagcagag ccagagcagg ttggagagga gacgcatcac ctccgctgct
     1981 cgcggggatc ctctagagtc gacggatccg gggaattcgc ggccgcactc gagatctagt
     2041 ggatcccccg ggctgcagga attatcacaa gtttgtacaa aaaagcaggc tccgcggccg
     2101 cccccttcac cagatctgct agcatgtccg tcctgacgcc gctgctgctg cggggcttga
     2161 caggctcggc ccggcggctc ccagtgccgc gcgccaagat ccattcgttg ggggatccac
     2221 cggtcgccac catggcctcc tccgagaacg tcatcaccga gttcatgcgc ttcaaggtgc
     2281 gcatggaggg caccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     2341 cctacgaggg ccacaacacc gtgaagctga aggtgaccaa gggcggcccc ctgcccttcg
     2401 cctgggacat cctgtccccc cagttccagt acggctccaa ggtgtacgtg aagcaccccg
     2461 ccgacatccc cgactacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     2521 tgaacttcga ggacggcggc gtggcgaccg tgacccagga ctcctccctg caggacggct
     2581 gcttcatcta caaggtgaag ttcatcggcg tgaacttccc ctccgacggc cccgtgatgc
     2641 agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc
     2701 tgaagggcga gatccacaag gccctgaagc tgaaggacgg cggccactac ctggtggagt
     2761 tcaagtccat ctacatggcc aagaagcccg tgcagctgcc cggctactac tacgtggaca
     2821 ccaagctgga catcacctcc cacaacgagg actacaccat cgtggagcag tacgagcgca
     2881 ccgagggccg ccaccacctg ttcctgtagc tcgagaaggg tgggcgcgcc gacccagctt
     2941 tcttgtacaa agtggtgata attcgatatc aagcttatcg ataccgtcga cctcgaggga
     3001 attccgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3061 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3121 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3181 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3241 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3301 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3361 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat
     3421 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3481 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3541 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     3601 gggaattcga gctcggtacc taatggcatt caccgcgtgc cttaattcga atggcattca
     3661 ccgcgtgcct taaacgcgtt ggcattcacc gcgtgcctta aatgcattgg cattcaccgc
     3721 gtgccttaag gtacctttaa gaccaatgac ttacaaggca gctgtagatc aattcaataa
     3781 aagatcttta ttttcattag atctgtgtgt tggttttttg tgtgcggccg caggaacccc
     3841 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     3901 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     3961 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4021 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     4081 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     4141 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4201 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4261 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4321 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     4381 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     4441 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     4501 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     4561 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4621 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4681 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     4741 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4801 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4861 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4921 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4981 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5041 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5101 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5161 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5221 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5281 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5341 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     5401 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     5461 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     5521 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     5581 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     5641 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     5701 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     5761 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5821 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5881 ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
