LOCUS       sequence-482468-        4234 bp    DNA     linear   UNA 25-AUG-2025
DEFINITION  Cre-dependent expression of FLAG-tagged Rpl22l1 and EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4234)
  AUTHORS   Geibl FF, Henrich MT, Xie Z, Zampese E, Ueda J, Tkatch T, Wokosin 
            DL, Nasiri E, Grotmann CA, Dawson VL, Dawson TM, Chandel NS, Oertel 
            WH, Surmeier DJ
  TITLE     alpha-Synuclein pathology disrupts mitochondrial function in 
            dopaminergic and cholinergic neurons at-risk in Parkinson's disease.
  JOURNAL   Mol Neurodegener. 2024 Oct 8;19(1):69. doi: 
            10.1186/s13024-024-00756-2.
  PUBMED    39379975
REFERENCE   2  (bases 1 to 4234)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol 
            Neurodegener."; date: "2024-10-8"; volume: "19(1):69. doi"; pages: "
            10.1186/s13024-024-00756-2"
COMMENT     Sequence Label: pFB-EF1a-DIO-Rpl22l1-3xFLAG-2A-GFP
FEATURES             Location/Qualifiers
     source          1..4234
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        231..1408
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1399
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1356..1376
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1439..1472)
                     /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(1523..1556)
                     /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(1565..2278,2279..2281,2282..2284))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1568..2278,2279..2281,2282..2284))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1610..1631)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1959..1978
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2218..2239
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2285..2338)
                     /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             complement(2351..2422)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     regulatory      2800..2809
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2816..2849
                     /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    2900..2933
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2958..3546
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3011..3031)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3429..3440)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3578..4054
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3777..3796)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4094..4234
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4094..4223
                     /label=AAV2 ITR
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc 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 acttccactg
      541 gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag
      601 gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg
      661 gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc
      721 tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct
      781 tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga
      841 cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc
      901 gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc
      961 gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc
     1021 ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc
     1081 gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt
     1141 cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag
     1201 cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca
     1261 cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga
     1321 atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag
     1381 tttttttctt ccatttcagg tgtcgtgagg taccggatcc tctagagtcg actccggaat
     1441 aacttcgtat aggatacttt atacgaagtt atgcagaatg gtagctggat tgtagctgct
     1501 attagcaata tgaaacctct taataacttc gtatagcata cattatacga agttatggcg
     1561 cgccttactt gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca
     1621 gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tgggtgctca
     1681 ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt
     1741 ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga
     1801 tgccgttctt ctgcttgtcg gccatgatat agacgttgtg gctgttgtag ttgtactcca
     1861 gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt
     1921 tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct
     1981 tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt
     2041 gctgcttcat gtggtcgggg tagcggctga agcactgcac gccgtaggtc agggtggtca
     2101 cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct
     2161 tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt
     2221 cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca
     2281 ccattgggcc aggattctcc tcgacgtcac cgcatgttag cagacttcct ctgccctctc
     2341 cactgccaga tttatcgtca tcatctttgt agtccttgtc atcatcgtcc ttatagtcct
     2401 tatcgtcgtc atccttgtaa tcgagcggcc gcgtacgcgt gtcctcagac tctgaaccat
     2461 cttcatcttg actaatctgg aaataacgaa gttcgtaagt ctccttgtca gatgcgacca
     2521 cacgtagcca atcacggaga ttattcttct taaggtattt cttggtaaga tatttcaaat
     2581 accttttgga gaactgtttc tcagaaacaa ctgtgatttt attcttcagg cgttcaatgt
     2641 gaacaacatt gccaagattt ccagtttttc cattgacttt aaccttctcc cgtagaaact
     2701 gttcaaaatt tccagagtca aaaattccat cttctactgg atgagtaagg tccaaatgaa
     2761 acctccaggt ggacttctta ggcttcttgt ctttcggcgc catggtggcg ctagcataac
     2821 ttcgtataaa gtatcctata cgaagttatt tgccttaacc cagaaattat cactgttatt
     2881 ctttagaatg gtgcaaagaa taacttcgta taatgtatgc tatacgaagt tatgaattcg
     2941 atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     3001 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     3061 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     3121 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     3181 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     3241 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3301 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     3361 cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct
     3421 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     3481 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     3541 ccccgcatcg ataccgagcg ctgctcgaga gatctacggg tggcatccct gtgacccctc
     3601 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat
     3661 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag
     3721 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta
     3781 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg
     3841 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac
     3901 caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct
     3961 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt
     4021 acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg
     4081 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4141 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4201 tcagtgagcg agcgagcgcg cagctgcctg cagg
//
