LOCUS       Exported                6556 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Targeted DNA methylation.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-5U6-sgRNAs-hsyn-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6556)
  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 6556)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6556
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        179..419
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        448..523
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        541..781
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        810..885
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        942..1182
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        1211..1286
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1304..1544
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        1573..1648
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1679..1919
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     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      2540..2549
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2546..3262
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2591..2612)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2852..2871)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3199..3220
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3303..3779
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3502..3521)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3819..3959
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3819..3948
                     /label=AAV2 ITR
     rep_origin      4034..4489
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4121..4140)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4331..4352
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4506..4525)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4625..4647
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4685..4703)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4771..4875
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4876..5736
                     /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(5094..5113)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5907..6495
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6396..6415
                     /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 gacgtagaaa
     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 gaaggtaccg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
     2581 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2641 cgagggcgag ggcgatgcca cctacggcaa gctgacacta aagttcatct gcaccaccgg
     2701 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     2761 cagtcgctat cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2821 ctacgtccag gagcgcacga tattctttaa ggacgacggc aactacaaga cccgcgccga
     2881 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgactttaa
     2941 agaagacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     3001 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     3061 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     3121 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     3181 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     3241 cggcatggac gagctgtaca agtgaaagct tatcgatacc gagcgctgct cgagagatct
     3301 acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc
     3361 cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt
     3421 ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga
     3481 caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt
     3541 ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt
     3601 tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac
     3661 ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg atctacccac
     3721 cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc
     3781 tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt
     3841 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     3901 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     3961 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     4021 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4081 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4141 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4201 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     4261 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4321 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat
     4381 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4441 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     4501 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4561 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4621 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4681 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4741 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4801 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4861 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4921 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4981 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5041 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5101 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5161 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5221 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5281 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5341 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5401 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5461 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5521 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5581 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5641 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5701 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     5761 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5821 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5881 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5941 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6001 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     6061 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6121 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6181 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6241 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6301 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6361 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6421 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6481 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6541 ccttttgctc acatgt
//
