LOCUS       sequence-217575-        6559 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeted DNA methylation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6559)
  AUTHORS   Lu Z, Liu Z, Mao W, Wang X, Zheng X, Chen S, Cao B, Huang S, Zhang 
            X, Zhou T, Zhang Y, Huang X, Sun Q, Li JD
  TITLE     Locus-specific DNA methylation of Mecp2 promoter leads to 
            autism-like phenotypes in mice.
  JOURNAL   Cell Death Dis. 2020 Feb 3;11(2):85. doi: 10.1038/s41419-020-2290-x.
  PUBMED    32015323
REFERENCE   2  (bases 1 to 6559)
  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: "Cell Death 
            Dis."; date: "2020-02-3"; volume: "11(2):85. doi"; pages: " 
            10.1038/s41419-020-2290-x"
COMMENT     Sequence Label: pAAV-5U6-sgRNAs-hsyn-EGFP
FEATURES             Location/Qualifiers
     source          1..6559
                     /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        179..427
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        448..523
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        541..789
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        810..885
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        942..1190
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1211..1286
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1304..1552
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1573..1648
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1679..1927
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1949..2024
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2079..2526
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      2543..2552
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2549..2551,2552..2554,2555..3268)
                     /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             join(2549..2551,2552..2554,2555..3265)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2594..2615)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2855..2874)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3202..3223
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3306..3782
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3505..3524)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3822..3962
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3822..3951
                     /label=AAV2 ITR
     rep_origin      4037..4492
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4124..4143)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4334..4355
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4509..4528)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4628..4650
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4688..4706)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4774..4878
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4879..4947,4948..5739)
                     /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(5097..5116)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5910..6498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6399..6418
                     /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 gcgtacgaga ctagcctcga gcggccgccc ccttcaccga
      181 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
      241 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
      301 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
      361 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
      421 aaacaccgca ggagttcctg tctgtttgtt ttagagctag aaatagcaag ttaaaataag
      481 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctagctcacc
      541 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
      601 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      661 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      721 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      781 cgaaacaccg cagcagcaca caggctggtg ttttagagct agaaatagca agttaaaata
      841 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttaaagggcc
      901 cgtcgacacg agactagcct cgagcggccg cccccttcac cgagggccta tttcccatga
      961 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
     1021 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     1081 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     1141 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gctccctcct
     1201 ctcggagaga gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     1261 ttgaaaaagt ggcaccgagt cggtgctttt tttctagctc accgagggcc tatttcccat
     1321 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
     1381 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     1441 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     1501 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgagtggga
     1561 ccgccaaggc cggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     1621 acttgaaaaa gtggcaccga gtcggtgctt tttttaaagg gcccgtcgac tgtctagcga
     1681 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     1741 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtataaa
     1801 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     1861 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
     1921 aaacaccggt ggagaaagcc accaccgggt tttagagcta gaaatagcaa gttaaaataa
     1981 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt taaagaattc
     2041 tcgacctcga gtctagactg cagagggccc tgcgtatgag tgcaagtggg ttttaggacc
     2101 aggatgaggc ggggtggggg tgcctacctg acgaccgacc ccgacccact ggacaagcac
     2161 ccaaccccca ttccccaaat tgcgcatccc ctatcagaga gggggagggg aaacaggatg
     2221 cggcgaggcg cgtgcgcact gccagcttca gcaccgcgga cagtgccttc gcccccgcct
     2281 ggcggcgcgc gccaccgccg cctcagcact gaaggcgcgc tgacgtcact cgccggtccc
     2341 ccgcaaactc cccttcccgg ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga
     2401 ccgcaccacg cgaggcgcga gatagggggg cacgggcgcg accatctgcg ctgcggcgcc
     2461 ggcgactcag cgctgcctca gtctgcggtg ggcagcggag gagtcgtgtc gtgcctgaga
     2521 gcgcagtcga gaaaccggta ccgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
     2581 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     2641 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     2701 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     2761 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2821 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2881 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2941 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     3001 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     3061 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     3121 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     3181 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     3241 tctcggcatg gacgagctgt acaagtgaaa gcttatcgat accgagcgct gctcgagaga
     3301 tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca
     3361 ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg
     3421 tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga
     3481 agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat
     3541 cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg
     3601 agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga
     3661 gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc
     3721 caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc
     3781 ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga
     3841 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     3901 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     3961 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     4021 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4081 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     4141 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4201 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4261 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4321 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     4381 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     4441 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     4501 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     4561 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4621 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     4681 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     4741 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     4801 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     4861 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     4921 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     4981 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5041 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5101 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5161 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5221 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5281 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5341 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5401 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5461 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5521 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5581 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5641 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     5701 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     5761 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     5821 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5881 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5941 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6001 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6061 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6121 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6181 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6241 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6301 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6361 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6421 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6481 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6541 tggccttttg ctcacatgt
//
