LOCUS       sequence-357624-        7382 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of mitochondria-targeted GCaMP6f under 
            control of the EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7382)
  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 7382)
  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-4mtGCaMP6f-DIO
FEATURES             Location/Qualifiers
     source          1..7382
                     /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(join(1593..2033,2034..2039,2040..2474,2475..2492,
                     2493..2762,2763..2768,2769..2825,2826..2852,2853..2876,
                     2877..2879,2880..2912,2913..2915,2916..2933,2934..2939))
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="GSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKT
                     GHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPV
                     LLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
                     FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
                     GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL
                     KGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRSL
                     GQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGNG
                     YISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     2149..2168
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2408..2429
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2535..2556)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2837..2846
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2970..3041)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             complement(3072..3143)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             complement(3174..3245)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             complement(3276..3350)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     protein_bind    3367..3400
                     /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    3451..3484
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3509..4097
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3562..3582)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3980..3991)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4129..4605
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4328..4347)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4645..4785
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4645..4774
                     /label=AAV2 ITR
     rep_origin      4860..5315
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4947..4966)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5157..5178
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5332..5351)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5451..5473
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5511..5529)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5597..5701
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5702..5770,5771..6562)
                     /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(5920..5939)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6733..7321
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7222..7241
                     /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 gctaatggcg cgccttatta ttaaccgtta tccttcgctg tcatcatttg tacaaactct
     1621 tcgtagttta cctgaccatc cccatcgatg tctgcttccc tgatcatttc atcaacctct
     1681 tcatctgtta acttctctcc aaggtttgtc atcacgtggc gaagctctgc tgcactgatg
     1741 tagccattgc catccttatc aaacacaccg aacgcttctc taatttcttc ttccgtgtcc
     1801 ctgtatttca tttttcttgc catcattgtc aggaactcag ggaagtcgat tgtgccgtca
     1861 ccgtcggcat ctacttcatt gatcatgtcc tgcagctctg cttctgtggg gttctgcccc
     1921 agagaccgca tcaccgtccc cagctccttg gttgttattg tcccatcccc gtccttgtca
     1981 aatagggaga attcctcttt aaattctgcg atctgctctt cagtcagttg gtccggcagg
     2041 ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc
     2101 tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg
     2161 ccgtcgtcct tgaagaagat ggtgcgctcc tggatgtagc cttcgggcat ggcggacttg
     2221 aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcactgcac gccgtaggtc
     2281 agggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc
     2341 agggtcagct tgccgtaggt ggcatcgccc tcaccctcgc cggacacgct gaacttgtgg
     2401 ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg
     2461 cccttgctca ccatgctccc tccggtaccg cccttgtaca gctcgtccat gccgagagtg
     2521 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct
     2581 ttcgaaagtt tggactgcac gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg
     2641 ccgatggggg tgttctgctg gtagtggtag gcgagctgca cgccgccgtc ctcgatgttg
     2701 tggcggatct tgaagttcgc cttgatgccg ttcttctgct tgtcggcctt gatatagacg
     2761 ttctcgagtg agctcagccg acctatagct ctgactgcgt gacctgtctt attccactta
     2821 cgacgtgatg agtcgaccat ggtggcgaga tccttatcgt catcgtcgta cagatcccga
     2881 cccatttgct gtccaccagt catgctagcc ataccatgat gatgatgatg atgagaacca
     2941 agcttcccct ccggcggcaa cgaatggatc ttggcgcgcg gcactgggag ccgccgggcc
     3001 gagcctgtca agccccgcag cagcagcggc gtcaggacgg acagtttccc ctccggcggc
     3061 aacgaatgga tcttggcgcg cggcactggg agccgccggg ccgagcctgt caagccccgc
     3121 agcagcagcg gcgtcaggac ggacagtttc ccctccggcg gcaacgaatg gatcttggcg
     3181 cgcggcactg ggagccgccg ggccgagcct gtcaagcccc gcagcagcag cggcgtcagg
     3241 acggacagtt tcccctccgg cggcaacgaa tggatcttgg cgcgcggcac tgggagccgc
     3301 cgggccgagc ctgtcaagcc ccgcagcagc agcggcgtca ggacggacat ggtgggctag
     3361 cctagcataa cttcgtataa agtatcctat acgaagttat ttgccttaac ccagaaatta
     3421 tcactgttat tctttagaat ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag
     3481 ttatgaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag
     3541 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     3601 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     3661 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     3721 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     3781 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     3841 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     3901 gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac
     3961 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     4021 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     4081 ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg gtggcatccc
     4141 tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc
     4201 cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt
     4261 atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc
     4321 tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc
     4381 tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag
     4441 gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata
     4501 ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa
     4561 ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta
     4621 accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     4681 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     4741 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     4801 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     4861 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4921 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4981 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5041 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     5101 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5161 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     5221 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5281 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     5341 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     5401 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5461 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     5521 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     5581 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5641 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5701 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5761 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5821 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5881 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5941 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6001 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6061 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6121 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6181 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6241 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6301 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6361 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6421 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6481 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6541 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6601 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6661 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6721 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6781 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6841 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6901 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6961 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7021 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7081 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7141 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7201 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7261 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7321 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7381 gt
//
