LOCUS Exported 5653 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION pAAV plasmid to induce expression of micro-RNA targeted against mouse Mecp2 and EGFP. ACCESSION . VERSION . KEYWORDS pAAV-CAG-GFP-mirMecp2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5653) 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 5653) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5653 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(11..28) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(182..770) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(262..281) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(941..1801) /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 1564..1583 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1802..1906) /gene="bla" /label=AmpR promoter rep_origin complement(1932..2387) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2069..2090) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2281..2300 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 2459..2588 /label=AAV2 ITR promoter 2899..3176 /label=chicken beta-actin promoter primer_bind 3455..3477 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 3539..3558 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" regulatory 3604..3613 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3610..4329 /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 3610..4326 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(3655..3676) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3916..3935) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4263..4284 /label=EGFP-C /note="EGFP, forward primer" ncRNA 4389..4415 /label=5' miR-155 /note="sequence upstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" ncRNA 4479..4520 /label=3' miR-155 /note="sequence downstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" misc_feature 4555..5143 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4608..4628) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5026..5037) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 5186..5307 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5197..5216) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 5251..5270 /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 5469..5598 /label=AAV2 ITR ORIGIN 1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt 2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag 2641 gaagagtacc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 2701 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 2761 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 2821 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 2881 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc 2941 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg 3001 gggcgggggg gggggggggg cgcgcgccrg gggggggggg ggggggsggg ggggggggsg 3061 gggsgrggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt 3121 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag 3181 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc 3241 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg 3301 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc 3361 ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg 3421 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 3481 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc 3541 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcc ccgggtaccg 3601 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 3661 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 3721 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 3781 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 3841 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 3901 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 3961 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 4021 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 4081 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 4141 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 4201 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 4261 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 4321 tacaagtaag ctaagcactt cgtggccgtc gatcgtttaa agggaggtag tgagtcgacc 4381 agtggatcct ggaggcttgc tgaaggctgt atgctgttca cctgaacacc ttctgatgtt 4441 ttggccactg actgacatca gaaggttcag gtgaacagga cacaaggcct gttactagca 4501 ctcacatgga acaaatggcc cagatctggc cgcactcgaa tcaagcttat cgataatcaa 4561 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt 4621 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct 4681 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc 4741 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg 4801 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc 4861 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc 4921 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt 4981 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca 5041 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt 5101 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgaccc 5161 gggcggccgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact 5221 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 5281 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag 5341 gttcaggggg agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa 5401 atcgataagg atcttcctag agcatggcta cgtagataag tagcatggcg ggttaatcat 5461 taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 5521 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 5581 gagcgagcga gcgcgcagct gcattaatga atcggccaac gcgcggggag aggcggtttg 5641 cgtattgggc gct //