LOCUS       Exported                6974 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Vector C encodes pAAV-pMecp2-dSaCas9-VP64-spA-pU6-sgRNA (BsaI) 
            transgenes for AAV packaging and expression of CRISPR activator in 
            neurons.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-pMecp2-dSaCas9-VP64-pU6-sgRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6974)
  AUTHORS   Lau CH, Ho JW, Lo PK, Tin C
  TITLE     Targeted Transgene Activation in the Brain Tissue by Systemic 
            Delivery of Engineered AAV1 Expressing CRISPRa.
  JOURNAL   Mol Ther Nucleic Acids. 2019 Jun 7;16:637-649. doi: 
            10.1016/j.omtn.2019.04.015. Epub 2019 Apr 23.
  PUBMED    31108320
REFERENCE   2  (bases 1 to 6974)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6974
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_RNA        96..171
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   185..325
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   185..314
                     /label=AAV2 ITR
     rep_origin      400..855
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(487..506)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     697..718
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(872..891)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     991..1013
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1051..1069)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1137..1241
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1242..2102
                     /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(1460..1479)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2273..2861
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2762..2781
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2923..3052
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        3071..3299
                     /label=Mecp2 promoter
                     /note="promoter for selective expression in neurons (Adachi
                     et al., 2005; Gray et al., 2011)"
     regulatory      3306..3315
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             3318..3338
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6519..6566
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6582..6731
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     polyA_signal    6747..6795
                     /note="synthetic polyadenylation signal"
     promoter        6801..67
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     6801..6821
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6972..17
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
       61 aaaggacgaa acaccggaga ccacggcagg tctcagtttt agtactctgg aaacagaatc
      121 tactaaaaca aggcaaaatg ccgtgtttat ctcgtcaact tgttggcgag atttttgcgg
      181 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
      241 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
      301 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg
      361 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
      421 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag
      481 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
      541 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
      601 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
      661 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
      721 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
      781 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
      841 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
      901 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
      961 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     1021 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     1081 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     1141 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1201 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1261 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1321 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1381 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1441 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     1501 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1561 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1621 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     1681 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     1741 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     1801 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     1861 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     1921 ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac
     1981 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2041 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2101 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2161 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2221 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2281 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2341 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2401 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2461 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2521 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2581 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2641 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2701 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2761 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     2821 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     2881 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg
     2941 ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
     3001 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggcctc
     3061 tagaaagctt agctgaatgg ggtccgcctc ttttccctgc ctaaacagac aggaactcct
     3121 gccaattgag ggcgtcaccg ctaaggctcc gccccagcct gggctccaca accaatgaag
     3181 ggtaatctcg acaaagagca aggggtgggg cgcgggcgcg caggtgcagc agcacacagg
     3241 ctggtcggga gggcggggcg cgacgtctgc cgtgcggggt cccggcatcg gttgcgcgca
     3301 ccggtgccac catggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag
     3361 ccaagcggaa ctacatcctg ggcctggcca tcggcatcac cagcgtgggc tacggcatca
     3421 tcgactacga gacacgggac gtgatcgatg ccggcgtgcg gctgttcaaa gaggccaacg
     3481 tggaaaacaa cgagggcagg cggagcaaga gaggcgccag aaggctgaag cggcggaggc
     3541 ggcatagaat ccagagagtg aagaagctgc tgttcgacta caacctgctg accgaccaca
     3601 gcgagctgag cggcatcaac ccctacgagg ccagagtgaa gggcctgagc cagaagctga
     3661 gcgaggaaga gttctctgcc gccctgctgc acctggccaa gagaagaggc gtgcacaacg
     3721 tgaacgaggt ggaagaggac accggcaacg agctgtccac caaagagcag atcagccgga
     3781 acagcaaggc cctggaagag aaatacgtgg ccgaactgca gctggaacgg ctgaagaaag
     3841 acggcgaagt gcggggcagc atcaacagat tcaagaccag cgactacgtg aaagaagcca
     3901 aacagctgct gaaggtgcag aaggcctacc accagctgga ccagagcttc atcgacacct
     3961 acatcgacct gctggaaacc cggcggacct actatgaggg acctggcgag ggcagcccct
     4021 tcggctggaa ggacatcaaa gaatggtacg agatgctgat gggccactgc acctacttcc
     4081 ccgaggaact gcggagcgtg aagtacgcct acaacgccga cctgtacaac gccctgaacg
     4141 acctgaacaa tctcgtgatc accagggacg agaacgagaa gctggaatat tacgagaagt
     4201 tccagatcat cgagaacgtg ttcaagcaga agaagaagcc caccctgaag cagatcgcca
     4261 aagaaatcct cgtgaacgaa gaggatatta agggctacag agtgaccagc accggcaagc
     4321 ccgagttcac caacctgaag gtgtaccacg acatcaagga cattaccgcc cggaaagaga
     4381 ttattgagaa cgccgagctg ctggatcaga ttgccaagat cctgaccatc taccagagca
     4441 gcgaggacat ccaggaagaa ctgaccaatc tgaactccga gctgacccag gaagagatcg
     4501 agcagatctc taatctgaag ggctataccg gcacccacaa cctgagcctg aaggccatca
     4561 acctgatcct ggacgagctg tggcacacca acgacaacca gatcgctatc ttcaaccggc
     4621 tgaagctggt gcccaagaag gtggacctgt cccagcagaa agagatcccc accaccctgg
     4681 tggacgactt catcctgagc cccgtcgtga agagaagctt catccagagc atcaaagtga
     4741 tcaacgccat catcaagaag tacggcctgc ccaacgacat cattatcgag ctggcccgcg
     4801 agaagaactc caaggacgcc cagaaaatga tcaacgagat gcagaagcgg aaccggcaga
     4861 ccaacgagcg gatcgaggaa atcatccgga ccaccggcaa agagaacgcc aagtacctga
     4921 tcgagaagat caagctgcac gacatgcagg aaggcaagtg cctgtacagc ctggaagcca
     4981 tccctctgga agatctgctg aacaacccct tcaactatga ggtggaccac atcatcccca
     5041 gaagcgtgtc cttcgacaac agcttcaaca acaaggtgct cgtgaagcag gaagaagcca
     5101 gcaagaaggg caaccggacc ccattccagt acctgagcag cagcgacagc aagatcagct
     5161 acgaaacctt caagaagcac atcctgaatc tggccaaggg caagggcaga atcagcaaga
     5221 ccaagaaaga gtatctgctg gaagaacggg acatcaacag gttctccgtg cagaaagact
     5281 tcatcaaccg gaacctggtg gataccagat acgccaccag aggcctgatg aacctgctgc
     5341 ggagctactt cagagtgaac aacctggacg tgaaagtgaa gtccatcaat ggcggcttca
     5401 ccagctttct gcggcggaag tggaagttta agaaagagcg gaacaagggg tacaagcacc
     5461 acgccgagga cgccctgatc attgccaacg ccgatttcat cttcaaagag tggaagaaac
     5521 tggacaaggc caaaaaagtg atggaaaacc agatgttcga ggaaaagcag gccgagagca
     5581 tgcccgagat cgaaaccgag caggagtaca aagagatctt catcaccccc caccagatca
     5641 agcacattaa ggacttcaag gactacaagt acagccaccg ggtggacaag aagcctaata
     5701 gagagctgat taacgacacc ctgtactcca cccggaagga cgacaagggc aacaccctga
     5761 tcgtgaacaa tctgaacggc ctgtacgaca aggacaatga caagctgaaa aagctgatca
     5821 acaagagccc cgaaaagctg ctgatgtacc accacgaccc ccagacctac cagaaactga
     5881 agctgattat ggaacagtac ggcgacgaga agaatcccct gtacaagtac tacgaggaaa
     5941 ccgggaacta cctgaccaag tactccaaaa aggacaacgg ccccgtgatc aagaagatta
     6001 agtattacgg caacaaactg aacgcccatc tggacatcac cgacgactac cccaacagca
     6061 gaaacaaggt cgtgaagctg tccctgaagc cctacagatt cgacgtgtac ctggacaatg
     6121 gcgtgtacaa gttcgtgacc gtgaagaatc tggatgtgat caaaaaagaa aactactacg
     6181 aagtgaatag caagtgctat gaggaagcta agaagctgaa gaagatcagc aaccaggccg
     6241 agtttatcgc ctccttctac aacaacgatc tgatcaagat caacggcgag ctgtatagag
     6301 tgatcggcgt gaacaacgac ctgctgaacc ggatcgaagt gaacatgatc gacatcacct
     6361 accgcgagta cctggaaaac atgaacgaca agaggccccc caggatcatt aagacaatcg
     6421 cctccaagac ccagagcatt aagaagtaca gcacagacat tctgggcaac ctgtatgaag
     6481 tgaaatctaa gaagcaccct cagatcatca aaaagggcaa aaggccggcg gccacgaaaa
     6541 aggccggcca ggcaaaaaag aaaaagggat ccggacgggc tgacgcattg gacgattttg
     6601 atctggatat gctgggaagt gacgccctcg atgattttga ccttgacatg cttggttcgg
     6661 atgcccttga tgactttgac ctcgacatgc tcggcagtga cgcccttgat gatttcgacc
     6721 tggacatgct gattaactaa gaattcaata aaagatcttt attttcatta gatctgtgtg
     6781 ttggtttttt gtgtggtacc gagggcctat ttcccatgat tccttcatat ttgcatatac
     6841 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     6901 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     6961 gttttaaaat ggac
//
