LOCUS       sequence-343518-        6459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of mitochondria-targeted Green Fluorescent 
            Protein under control of the EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6459)
  AUTHORS   Kole K, Voesenek BJB, Brinia ME, Petersen N, Kole MHP
  TITLE     Parvalbumin basket cell myelination accumulates axonal mitochondria 
            to internodes.
  JOURNAL   Nat Commun. 2022 Dec 9;13(1):7598. doi: 10.1038/s41467-022-35350-x.
  PUBMED    36494349
REFERENCE   2  (bases 1 to 6459)
  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: "2022-12-9"; volume: "13(1):7598. doi"; pages: " 
            10.1038/s41467-022-35350-x"
COMMENT     Sequence Label: pAAV-EF1a-mitoGFP-DIO
FEATURES             Location/Qualifiers
     source          1..6459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1440..1473)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1524..1557)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1580..2296)
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1623..1643)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     2238..2266
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     CDS             complement(2297..2323)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             complement(2360..2434)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     protein_bind    2444..2477
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2528..2561
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2586..3174
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2639..2659)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3057..3068)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3206..3682
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3405..3424)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3722..3862
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3722..3851
                     /label=AAV2 ITR
     rep_origin      3937..4392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4024..4043)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4234..4255
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4409..4428)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4528..4550
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4588..4606)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4674..4778
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4779..4847,4848..5639)
                     /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     complement(4997..5016)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5810..6398
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6299..6318
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
     1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1561 gcgccggaag tctggacatt tatttgtata gttcatccat gccatgtgta atcccagcag
     1621 ctgttacaaa ctcaagaagg accatgtggt ctctcttttc gttgggatct ttcgaaaggg
     1681 cagattgtgt ggacaggtaa tggttgtctg gtaaaaggac agggccatcg ccaattggag
     1741 tattttgttg ataatgatct gctagttgaa cgcttccatc ttcaatgttg tgtctaattt
     1801 tgaagttaac tttgattcca ttcttttgtt tgtctgccat gatgtataca ttgtgtgagt
     1861 tatagttgta ttccaatttg tgtccaagaa tgtttccatc ttctttaaaa tcaatacctt
     1921 ttaactcgat tctattaaca agggtatcac cttcaaactt gacttcagca cgtgtcttgt
     1981 agttcccgtc atctttgaaa aatatagttc tttcctgtac ataaccttcg ggcatggcac
     2041 tcttgaaaaa gtcatgccgt ttcatatgat ctgggtatct tgaaaagcat tgaacaccat
     2101 aagagaaagt agtgacaagt gttggccatg gaacaggtag ttttccagta gtgcaaataa
     2161 atttaagggt aagttttccg tatgttgcat caccttcacc ctctccactg acagaaaatt
     2221 tgtgcccatt aacatcacca tctaattcaa caagaattgg gacaactcca gtgaaaagtt
     2281 cttctccttt actcattaag cttgcataat caggaacatc ataatcgata agcttcccct
     2341 ccggcggcaa cgaatggatc ttggcgcgcg gcactgggag ccgccgggcc gagcctgtca
     2401 agccccgcag cagcagcggc gtcaggacgg acatgatgct agcataactt cgtataaagt
     2461 atcctatacg aagttatttg ccttaaccca gaaattatca ctgttattct ttagaatggt
     2521 gcaaagaata acttcgtata atgtatgcta tacgaagtta tgaattcgat atcaagctta
     2581 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     2641 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     2701 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     2761 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     2821 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     2881 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     2941 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     3001 tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     3061 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     3121 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     3181 accgagcgct gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct
     3241 cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg
     3301 catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg
     3361 gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag
     3421 ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat
     3481 tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta
     3541 atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc
     3601 ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac
     3661 cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg
     3721 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     3781 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     3841 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     3901 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     3961 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     4021 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4081 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4141 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4201 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4261 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     4321 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4381 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4441 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4501 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4561 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     4621 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4681 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4741 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4801 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4861 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4921 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4981 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5041 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5101 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5161 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5221 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5281 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5341 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5401 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     5461 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     5521 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     5581 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     5641 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5701 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5761 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5821 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5881 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5941 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6001 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6061 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6121 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6181 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6241 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6301 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6361 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6421 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
