LOCUS       Exported                7033 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Vector E encodes pAAV-pMecp2-dSaCas9-KRAB-spA-pU6-sgRNA (BsaI) 
            transgenes for AAV packaging and expression of CRISPR interference 
            in neurons.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-pMecp2-dSaCas9-KRAB-pU6-sgRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7033)
  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 7033)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7033
                     /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        149..377
                     /label=Mecp2 promoter
                     /note="promoter for selective expression in neurons (Adachi
                     et al., 2005; Gray et al., 2011)"
     regulatory      384..393
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             396..416
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3596..3643
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3680..3874
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     polyA_signal    3884..3932
                     /note="synthetic polyadenylation signal"
     promoter        3938..4178
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     3938..3958
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4109..4128
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4207..4282
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4296..4436
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4296..4425
                     /label=AAV2 ITR
     rep_origin      4511..4966
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4598..4617)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4808..4829
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4983..5002)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5102..5124
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5162..5180)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5248..5352
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5353..6213
                     /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(5571..5590)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6384..6972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6873..6892
                     /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 gcggcctcta gaaagcttag ctgaatgggg tccgcctctt ttccctgcct
      181 aaacagacag gaactcctgc caattgaggg cgtcaccgct aaggctccgc cccagcctgg
      241 gctccacaac caatgaaggg taatctcgac aaagagcaag gggtggggcg cgggcgcgca
      301 ggtgcagcag cacacaggct ggtcgggagg gcggggcgcg acgtctgccg tgcggggtcc
      361 cggcatcggt tgcgcgcacc ggtgccacca tggccccaaa gaagaagcgg aaggtcggta
      421 tccacggagt cccagcagcc aagcggaact acatcctggg cctggccatc ggcatcacca
      481 gcgtgggcta cggcatcatc gactacgaga cacgggacgt gatcgatgcc ggcgtgcggc
      541 tgttcaaaga ggccaacgtg gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa
      601 ggctgaagcg gcggaggcgg catagaatcc agagagtgaa gaagctgctg ttcgactaca
      661 acctgctgac cgaccacagc gagctgagcg gcatcaaccc ctacgaggcc agagtgaagg
      721 gcctgagcca gaagctgagc gaggaagagt tctctgccgc cctgctgcac ctggccaaga
      781 gaagaggcgt gcacaacgtg aacgaggtgg aagaggacac cggcaacgag ctgtccacca
      841 aagagcagat cagccggaac agcaaggccc tggaagagaa atacgtggcc gaactgcagc
      901 tggaacggct gaagaaagac ggcgaagtgc ggggcagcat caacagattc aagaccagcg
      961 actacgtgaa agaagccaaa cagctgctga aggtgcagaa ggcctaccac cagctggacc
     1021 agagcttcat cgacacctac atcgacctgc tggaaacccg gcggacctac tatgagggac
     1081 ctggcgaggg cagccccttc ggctggaagg acatcaaaga atggtacgag atgctgatgg
     1141 gccactgcac ctacttcccc gaggaactgc ggagcgtgaa gtacgcctac aacgccgacc
     1201 tgtacaacgc cctgaacgac ctgaacaatc tcgtgatcac cagggacgag aacgagaagc
     1261 tggaatatta cgagaagttc cagatcatcg agaacgtgtt caagcagaag aagaagccca
     1321 ccctgaagca gatcccaaag aaatcctcgt gaacgaagag gatattaagg gctacagagt
     1381 gaccagcacc ggcaagcccg agttcaccaa cctgaaggtg taccacgaca tcaaggacat
     1441 taccgcccgg aaagagatta ttgagaacgc cgagctgctg gatcagattg ccaagatcct
     1501 gaccatctac cagagcagcg aggacatcca ggaagaactg accaatctga actccgagct
     1561 gacccaggaa gagatcgagc agatctctaa tctgaagggc tataccggca cccacaacct
     1621 gagcctgaag gccatcaacc tgatcctgga cgagctgtgg cacaccaacg acaaccagat
     1681 cgctatcttc aaccggctga agctggtgcc caagaaggtg gacctgtccc agcagaaaga
     1741 gatccccacc accctggtgg acgacttcat cctgagcccc gtcgtgaaga gaagcttcat
     1801 ccagagcatc aaagtgatca acgccatcat caagaagtac ggcctgccca acgacatcat
     1861 tatcgagctg gcccgcgaga agaactccaa ggacgcccag aaaatgatca acgagatgca
     1921 gaagcggaac cggcagacca acgagcggat cgaggaaatc atccggacca ccggcaaaga
     1981 gaacgccaag tacctgatcg agaagatcaa gctgcacgac atgcaggaag gcaagtgcct
     2041 gtacagcctg gaagccatcc ctctggaaga tctgctgaac aaccccttca actatgaggt
     2101 ggaccacatc atccccagaa gcgtgtcctt cgacaacagc ttcaacaaca aggtgctcgt
     2161 gaagcaggaa gaagccagca agaagggcaa ccggacccca ttccagtacc tgagcagcag
     2221 cgacagcaag atcagctacg aaaccttcaa gaagcacatc ctgaatctgg ccaagggcaa
     2281 gggcagaatc agcaagacca agaaagagta tctgctggaa gaacgggaca tcaacaggtt
     2341 ctccgtgcag aaagacttca tcaaccggaa cctggtggat accagatacg ccaccagagg
     2401 cctgatgaac ctgctgcgga gctacttcag agtgaacaac ctggacgtga aagtgaagtc
     2461 catcaatggc ggcttcacca gctttctgcg gcggaagtgg aagtttaaga aagagcggaa
     2521 caaggggtac aagcaccacg ccgaggacgc cctgatcatt gccaacgccg atttcatctt
     2581 caaagagtgg aagaaactgg acaaggccaa aaaagtgatg gaaaaccaga tgttcgagga
     2641 aaagcaggcc gagagcatgc ccgagatcga aaccgagcag gagtacaaag agatcttcat
     2701 caccccccac cagatcaagc acattaagga cttcaaggac tacaagtaca gccaccgggt
     2761 ggacaagaag cctaatagag agctgattaa cgacaccctg tactccaccc ggaaggacga
     2821 caagggcaac accctgatcg tgaacaatct gaacggcctg tacgacaagg acaatgacaa
     2881 gctgaaaaag ctgatcaaca agagccccga aaagctgctg atgtaccacc acgaccccca
     2941 gacctaccag aaactgaagc tgattatgga acagtacggc gacgagaaga atcccctgta
     3001 caagtactac gaggaaaccg ggaactacct gaccaagtac tccaaaaagg acaacggccc
     3061 cgtgatcaag aagattaagt attacggcaa caaactgaac gcccatctgg acatcaccga
     3121 cgactacccc aacagcagaa acaaggtcgt gaagctgtcc ctgaagccct acagattcga
     3181 cgtgtacctg gacaatggcg tgtacaagtt cgtgaccgtg aagaatctgg atgtgatcaa
     3241 aaaagaaaac tactacgaag tgaatagcaa gtgctatgag gaagctaaga agctgaagaa
     3301 gatcagcaac caggccgagt ttatcgcctc cttctacaac aacgatctga tcaagatcaa
     3361 cggcgagctg tatagagtga tcggcgtgaa caacgacctg ctgaaccgga tcgaagtgaa
     3421 catgatcgac atcacctacc gcgagtacct ggaaaacatg aacgacaaga ggccccccag
     3481 gatcattaag acaatcgcct ccaagaccca gagcattaag aagtacagca cagacattct
     3541 gggcaacctg tatgaagtga aatctaagaa gcaccctcag atcatcaaaa agggcaaaag
     3601 gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagggatcca tggacgcgaa
     3661 atcacttacg gcatggtcga gaacactggt tacgttcaag gacgtgtttg tggactttac
     3721 acgtgaggag tggaaattgc tggatactgc gcaacaaatt gtgtatcgaa atgtcatgct
     3781 tgagaattac aagaacctcg tcagtctcgg ataccagttg acgaaaccgg atgtgatcct
     3841 taggctcgaa aagggggaag aaccttggct ggtataagaa ttcaataaaa gatctttatt
     3901 ttcattagat ctgtgtgttg gttttttgtg tggtaccgag ggcctatttc ccatgattcc
     3961 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
     4021 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     4081 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     4141 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggag accacggcag
     4201 gtctcagttt tagtactctg gaaacagaat ctactaaaac aaggcaaaat gccgtgttta
     4261 tctcgtcaac ttgttggcga gatttttgcg gccgcaggaa cccctagtga tggagttggc
     4321 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     4381 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     4441 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4501 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4561 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     4621 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4681 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     4741 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4801 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     4861 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     4921 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     4981 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     5041 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5101 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     5161 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5221 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5281 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5341 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5401 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5461 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5521 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5581 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5641 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5701 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5761 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5821 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5881 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5941 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6001 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6061 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6121 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6181 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6241 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6301 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6361 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6421 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6481 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6541 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6601 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6661 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6721 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6781 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6841 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6901 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6961 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7021 tttgctcaca tgt
//
