LOCUS       sequence-327851-        7039 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAAV plasmid for Cre-dependent expression of human MECP2 fused with 
            cycle3 GFP under Syn promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7039)
  AUTHORS   Ito-Ishida A, Baker SA, Sillitoe RV, Sun Y, Zhou J, Ono Y, Iwakiri 
            J, Yuzaki M, Zoghbi HY
  TITLE     MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in 
            Mouse Neurons.
  JOURNAL   J Neurosci. 2020 Nov 4;40(45):8746-8766. doi: 
            10.1523/JNEUROSCI.1281-19.2020. Epub 2020 Oct 12.
  PUBMED    33046553
REFERENCE   2  (bases 1 to 7039)
  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"; doi: 
            "10.1523/JNEUROSCI.1281-19.2020"; journalName: "J Neurosci"; date: 
            "2020-11-4- 4"; volume: "40"; issue: "45"; pages: "8746-8766"
COMMENT     Sequence Label: pAAV-hSyn-DIO-hMECP2-cycle3GFP
FEATURES             Location/Qualifiers
     source          1..7039
                     /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        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(660..693)
                     /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(744..777)
                     /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(804..1517,1518..1520,1521..1523))
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(847..867)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     1462..1490
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     protein_bind    1534..1558
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1899..1919)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     protein_bind    3024..3057
                     /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    3108..3141
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3166..3754
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3219..3239)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3637..3648)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3786..4262
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3985..4004)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4302..4442
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4302..4431
                     /label=AAV2 ITR
     rep_origin      4517..4972
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4604..4623)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4814..4835
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4989..5008)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5108..5130
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5168..5186)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5254..5358
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5359..5427,5428..6219)
                     /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(5577..5596)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6390..6978
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6879..6898
                     /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 gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc gactccggaa
      661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
      721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
      781 gcgccactag tcgatccgcc gttttatttg tagagctcat ccatgccatg tgtaatccca
      841 gcagcagtta caaactcaag aaggaccatg tggtcacgct tttcgttggg atctttcgaa
      901 agggcagatt gtgtcgacag gtaatggttg tctggtaaaa ggacagggcc atcgccaatt
      961 ggagtatttt gttgataatg gtctgctagt tgaacggatc catcttcaat gttgtggcga
     1021 attttgaagt tagctttgat tccattcttt tgtttgtctg ccgtgatgta tacattgtgt
     1081 gagttatagt tgtactcgag tttgtgtccg agaatgtttc catcttcttt aaaatcaata
     1141 ccttttaact cgatacgatt aacaagggta tcaccttcaa acttgacttc agcacgcgtc
     1201 ttgtagttcc cgtcatcttt gaaagatata gtgcgttcct gtacataacc ttcgggcatg
     1261 gcactcttga aaaagtcatg ccgtttcata tgatccggat aacgggaaaa gcattgaaca
     1321 ccataagaga aagtagtgac aagtgttggc catggaacag gtagttttcc agtagtgcaa
     1381 ataaatttaa gggtaagctt tccgtatgta gcatcacctt caccctctcc actgacagaa
     1441 aatttgtgcc cattaacatc accatctaat tcaacaagaa ttgggacaac tccagtgaaa
     1501 agttcttctc ctttgctagc cattctagat cgaaccactt tgtacaagaa agttgggcta
     1561 actctctcgg tcacgggcgt ccggctgtcc acaggctcct ctctgtttgg ccttggcatg
     1621 gaggatgaaa caatgtcttt gcgctctccc tcccctcggt gtttgtactt ttctgcggcc
     1681 gtggcggcgg tggcaaccgc gggctgagtc ttagctggct ccttggggca gccgtcgctc
     1741 tccagtgagc ctcctctggg catcttctcc tctttgcaga cgctgctgct caagtcctgg
     1801 ggctcagggg ggctggtggg gtcctcggag ctctcgggct caggtggagg tgggggcagg
     1861 ggtgggagca gtggcacggg ggcctttggg gactctgagt ggtggtgatg gtggtggtgc
     1921 tccttcttgg ggggtgagga ggcgctgctg ctgcgcccct tggggctgct ctccttgctt
     1981 ttccgcccag ggctcttaca ggtcttcagt cctttcccgc tcttctcacc gagggtggac
     2041 accagcaggg gcttcaccac ttccttgacc tcgatgctga ccgtctcccg ggtcttgcgc
     2101 ttcttgatgg ggagtacggt ctcctgcaca gatcggatag aagactcctt cacggctttc
     2161 tttttggcct cggcggcagc ggctgccacc acactccccg gctttcggcc ccgtttcttg
     2221 ggaatggcct gagggtcggc ctcagctttt cgcttcctgc cggggcgttt gatcaccatg
     2281 acctgggtgg atgtggtggc cccacccccc tcagccttgc cccctggcga agtttgaaaa
     2341 ggcatcttga caaggagctt cccaggactt ttctccagga cccttttcac ctgcacaccc
     2401 tctgacgtgg ccgccttggg tctcgtggtg ccgctccctt tggggcgtcc ccggcctctg
     2461 ccagttcctg gagctttggg agatttgggc ttcttaggtg gtttctgctc tcgccgggag
     2521 gggctccctc tcccagttac cgtgaagtca aaatcattag ggtccaggga tgtgtcgcct
     2581 accttttcga agtacgcaat caactccact ttagagcgaa aggcttttcc ctggggattg
     2641 atcaaataca catcatactt cccagcagag cggccagatt tcctttgctt aagcttccgt
     2701 gtccagcctt caggcagggt ggggtcatca tacatgggtc cccggtcacg gatgatggag
     2761 cgccgctgtt tgggggaggc agaagcttcc ggcacagccg gggcggagcc tgacccttct
     2821 gatgtctctg ctttgcctgc ctctgcgggc tcagcagagt ggtgggctga tggctgcacg
     2881 ggctcatgct tgccctcttt ctcttctttc ttatctttct tcaccttttt aaacttgagg
     2941 ggtttgtcct tgaggccctg gaggtcctgg tcttctgact tttcttccct gagccctaac
     3001 atcccagcta ccataacgct agcataactt cgtataaagt atcctatacg aagttatttg
     3061 ccttaaccca gaaattatca ctgttattct ttagaatggt gcaaagaata acttcgtata
     3121 atgtatgcta tacgaagtta tgaattcgat atcaagctta tcgataatca acctctggat
     3181 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     3241 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     3301 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     3361 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     3421 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     3481 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3541 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc
     3601 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3661 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     3721 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga
     3781 tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca
     3841 ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg
     3901 tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga
     3961 agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat
     4021 cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg
     4081 agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga
     4141 gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc
     4201 caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc
     4261 ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga
     4321 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     4381 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     4441 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     4501 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4561 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     4621 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4681 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4741 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4801 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     4861 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     4921 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     4981 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     5041 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5101 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     5161 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     5221 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5281 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5341 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5401 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5461 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5521 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5581 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5641 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5701 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5761 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5821 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5881 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5941 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6001 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6061 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6121 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6181 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6241 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6301 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6361 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6421 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6481 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6541 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6601 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6661 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6721 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6781 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6841 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6901 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6961 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7021 tggccttttg ctcacatgt
//
