LOCUS       PX552-CMV-TD-V2         5591 bp ds-DNA     circular     18-JAN-2022
DEFINITION  .
KEYWORDS    "creator:Zhang Lab / Addgene #60958" "marker:AmpR, AAV2 ITR, AAV2
            ITR" "organism:Mammalian Cells" "ref:pmid\:25326897"
COMMENT     Zhang lab AAV vector, also known as AAV-SpGuide or pAAV SpCas9
            acceptor, for sgRNA expression with a nuclear EGFP marker. Use
            together with PX551. Digest with SapI to insert annealed oligos
            encoding the guide sequence. <small>[1] In vivo interrogation of
            gene function in the mammalian brain using CRISPR-Cas9. Nat.
            Biotechnol. 2015;33:102-6. Swiech L, Heidenreich M, Banerjee A,
            Habib N, Li Y, Trombetta J, Sur M, Zhang F. (pmid:25326897)</small>
FEATURES             Location/Qualifiers
     repeat_region   1..141
                     /label="AAV2 ITR"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
     primer          156..175
                     /label="U6FWD-Seq"
                     /note="sequence: GAGGGCCTATTTCCCATGAT"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          156..177
                     /label="px552_insert-FWDwith PspOMI"
                     /note="sequence: GAGGGCCTATTTCCCATGATTC"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     promoter        156..396
                     /label="U6 promoter"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     misc_feature    405..424
                     /label="px552-Tandem V2 site (mu/hu/mk)"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     misc_RNA        426..501
                     /label="gRNA scaffold"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     primer          complement(496..520)
                     /label="px552_insert_RVS with PspOMI"
                     /note="sequence: TCTGCAGTCTAGAAAAAAAGCACCG"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     terminator      502..507
                     /label="pol III terminator"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     misc_feature    517..521
                     /label="PspOMI RE Site"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     primer          complement(543..570)
                     /label="Seq-px552 tandem-RVS"
                     /note="sequence: CGCCTAACCCTAAGCAGATTCTTCATGC"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
     primer          complement(586..608)
                     /label="Seq-px552 tandem-RVS2"
                     /note="sequence: GCGTATATCTGGCCCGTACATCG"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     enhancer        612..991
                     /label="CMV enhancer"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     primer          complement(650..672)
                     /label="Seq-px552 tandem-RVS3"
                     /note="sequence: GGGCTATGAACTAATGACCCCGT"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     promoter        993..1191
                     /label="CMV promoter"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     misc_feature    complement(1286..1293)
                     /label="BamHI RE Site"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
     CDS             1305..1307
                     /label="EGFP"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     CDS             1308..1310
                     /label="EGFP"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     CDS             1311..2021
                     /label="EGFP"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     CDS             2040..2288
                     /label="KASH"
                     /ApEinfo_revcolor="#993366"
                     /ApEinfo_fwdcolor="#993366"
     polyA_signal    2338..2814
                     /label="hGH poly(A) signal"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     repeat_region   2854..2994
                     /label="AAV2 ITR"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
     rep_origin      3069..3524
                     /label="f1 ori"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     promoter        3806..3910
                     /label="AmpR promoter"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     CDS             3911..3979
                     /label="AmpR"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     CDS             3980..4771
                     /label="AmpR"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     rep_origin      4942..5530
                     /label="ori"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
ORIGIN
        1 CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC
       61 GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA
      121 ACTCCATCAC TAGGGGTTCC TGCGGCCGCA CGCGTGAGGG CCTATTTCCC ATGATTCCTT
      181 CATATTTGCA TATACGATAC AAGGCTGTTA GAGAGATAAT TGGAATTAAT TTGACTGTAA
      241 ACACAAAGAT ATTAGTACAA AATACGTGAC GTAGAAAGTA ATAATTTCTT GGGTAGTTTG
      301 CAGTTTTAAA ATTATGTTTT AAAATGGACT ATCATATGCT TACCGTAACT TGAAAGTATT
      361 TCGATTTCTT GGCTTTATAT ATCTTGTGGA AAGGACGAAA CACCGAGAAG TGCTAGCTCG
      421 GGCCTGTTTT AGAGCTAGAA ATAGCAAGTT AAAATAAGGC TAGTCCGTTA TCAACTTGAA
      481 AAAGTGGCAC CGAGTCGGTG CTTTTTTTCT AGACTGCAGA GGGCCCGGCT TGACCGACAA
      541 TTGCATGAAG AATCTGCTTA GGGTTAGGCG TTTTGCGCTG CTTCGCGATG TACGGGCCAG
      601 ATATACGCGT TGACATTGAT TATTGACTAG TTATTAATAG TAATCAATTA CGGGGTCATT
      661 AGTTCATAGC CCATATATGG AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGG
      721 CTGACCGCCC AACGACCCCC GCCCATTGAC GTCAATAATG ACGTATGTTC CCATAGTAAC
      781 GCCAATAGGG ACTTTCCATT GACGTCAATG GGTGGAGTAT TTACGGTAAA CTGCCCACTT
      841 GGCAGTACAT CAAGTGTATC ATATGCCAAG TACGCCCCCT ATTGACGTCA ATGACGGTAA
      901 ATGGCCCGCC TGGCATTATG CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA
      961 CATCTACGTA TTAGTCATCG CTATTACCAT GGTGATGCGG TTTTGGCAGT ACATCAATGG
     1021 GCGTGGATAG CGGTTTGACT CACGGGGATT TCCAAGTCTC CACCCCATTG ACGTCAATGG
     1081 GAGTTTGTTT TGGCACCAAA ATCAACGGGA CTTTCCAAAA TGTCGTAACA ACTCCGCCCC
     1141 ATTGACGCAA ATGGGCGGTA GGCGTGTACG GTGGGAGGTC TATATAAGCA GCGCGTTTTG
     1201 CCTGTACTGG GTCTCTCTGG TTAGACCAGA TCTGAGCCTG GGAGCTCTCT GGCTAACTAG
     1261 GGAACCCACT GCTTAAGCCG GATCCCTCTA GAGTCGACGC CACCATGGTG AGCAAGGGCG
     1321 AGGAGCTGTT CACCGGGGTG GTGCCCATCC TGGTCGAGCT GGACGGCGAC GTAAACGGCC
     1381 ACAAGTTCAG CGTGTCCGGC GAGGGCGAGG GCGATGCCAC CTACGGCAAG CTGACCCTGA
     1441 AGTTCATCTG CACCACCGGC AAGCTGCCCG TGCCCTGGCC CACCCTCGTG ACCACCCTGA
     1501 CCTACGGCGT GCAGTGCTTC AGCCGCTACC CCGACCACAT GAAGCAGCAC GACTTCTTCA
     1561 AGTCCGCCAT GCCCGAAGGC TACGTCCAGG AGCGCACCAT CTTCTTCAAG GACGACGGCA
     1621 ACTACAAGAC CCGCGCCGAG GTGAAGTTCG AGGGCGACAC CCTGGTGAAC CGCATCGAGC
     1681 TGAAGGGCAT CGACTTCAAG GAGGACGGCA ACATCCTGGG GCACAAGCTG GAGTACAACT
     1741 ACAACAGCCA CAACGTCTAT ATCATGGCCG ACAAGCAGAA GAACGGCATC AAGGTGAACT
     1801 TCAAGATCCG CCACAACATC GAGGACGGCA GCGTGCAGCT CGCCGACCAC TACCAGCAGA
     1861 ACACCCCCAT CGGCGACGGC CCCGTGCTGC TGCCCGACAA CCACTACCTG AGCACCCAGT
     1921 CCGCCCTGAG CAAAGACCCC AACGAGAAGC GCGATCACAT GGTCCTGCTG GAGTTCGTGA
     1981 CCGCCGCCGG GATCACTCTC GGCATGGACG AGCTGTACAA GTCCGGACTC AGATCTCGAG
     2041 AGGAGGAGGA GGAGACAGAC AGCAGGATGC CCCACCTCGA CAGCCCCGGC AGCTCCCAGC
     2101 CGAGACGCTC CTTCCTCTCA AGGGTGATCA GGGCAGCGCT ACCGTTGCAG CTGCTTCTGC
     2161 TGCTGCTGCT GCTCCTGGCC TGCCTGCTAC CTGCCTCTGA AGATGACTAC AGCTGCACCC
     2221 AGGCCAACAA CTTTGCCCGA TCCTTCTACC CCATGCTGCG GTACACCAAC GGGCCACCTC
     2281 CCACCTAGGA ATTCGATATC AAGCTTATCG ATACCGAGCG CTGCTCGAGA GATCTACGGG
     2341 TGGCATCCCT GTGACCCCTC CCCAGTGCCT CTCCTGGCCC TGGAAGTTGC CACTCCAGTG
     2401 CCCACCAGCC TTGTCCTAAT AAAATTAAGT TGCATCATTT TGTCTGACTA GGTGTCCTTC
     2461 TATAATATTA TGGGGTGGAG GGGGGTGGTA TGGAGCAAGG GGCAAGTTGG GAAGACAACC
     2521 TGTAGGGCCT GCGGGGTCTA TTGGGAACCA AGCTGGAGTG CAGTGGCACA ATCTTGGCTC
     2581 ACTGCAATCT CCGCCTCCTG GGTTCAAGCG ATTCTCCTGC CTCAGCCTCC CGAGTTGTTG
     2641 GGATTCCAGG CATGCATGAC CAGGCTCAGC TAATTTTTGT TTTTTTGGTA GAGACGGGGT
     2701 TTCACCATAT TGGCCAGGCT GGTCTCCAAC TCCTAATCTC AGGTGATCTA CCCACCTTGG
     2761 CCTCCCAAAT TGCTGGGATT ACAGGCGTGA ACCACTGCTC CCTTCCCTGT CCTTCTGATT
     2821 TTGTAGGTAA CCACGTGCGG ACCGAGCGGC CGCAGGAACC CCTAGTGATG GAGTTGGCCA
     2881 CTCCCTCTCT GCGCGCTCGC TCGCTCACTG AGGCCGGGCG ACCAAAGGTC GCCCGACGCC
     2941 CGGGCTTTGC CCGGGCGGCC TCAGTGAGCG AGCGAGCGCG CAGCTGCCTG CAGGGGCGCC
     3001 TGATGCGGTA TTTTCTCCTT ACGCATCTGT GCGGTATTTC ACACCGCATA CGTCAAAGCA
     3061 ACCATAGTAC GCGCCCTGTA GCGGCGCATT AAGCGCGGCG GGTGTGGTGG TTACGCGCAG
     3121 CGTGACCGCT ACACTTGCCA GCGCCCTAGC GCCCGCTCCT TTCGCTTTCT TCCCTTCCTT
     3181 TCTCGCCACG TTCGCCGGCT TTCCCCGTCA AGCTCTAAAT CGGGGGCTCC CTTTAGGGTT
     3241 CCGATTTAGT GCTTTACGGC ACCTCGACCC CAAAAAACTT GATTTGGGTG ATGGTTCACG
     3301 TAGTGGGCCA TCGCCCTGAT AGACGGTTTT TCGCCCTTTG ACGTTGGAGT CCACGTTCTT
     3361 TAATAGTGGA CTCTTGTTCC AAACTGGAAC AACACTCAAC CCTATCTCGG GCTATTCTTT
     3421 TGATTTATAA GGGATTTTGC CGATTTCGGC CTATTGGTTA AAAAATGAGC TGATTTAACA
     3481 AAAATTTAAC GCGAATTTTA ACAAAATATT AACGTTTACA ATTTTATGGT GCACTCTCAG
     3541 TACAATCTGC TCTGATGCCG CATAGTTAAG CCAGCCCCGA CACCCGCCAA CACCCGCTGA
     3601 CGCGCCCTGA CGGGCTTGTC TGCTCCCGGC ATCCGCTTAC AGACAAGCTG TGACCGTCTC
     3661 CGGGAGCTGC ATGTGTCAGA GGTTTTCACC GTCATCACCG AAACGCGCGA GACGAAAGGG
     3721 CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA ATAATGGTTT CTTAGACGTC
     3781 AGGTGGCACT TTTCGGGGAA ATGTGCGCGG AACCCCTATT TGTTTATTTT TCTAAATACA
     3841 TTCAAATATG TATCCGCTCA TGAGACAATA ACCCTGATAA ATGCTTCAAT AATATTGAAA
     3901 AAGGAAGAGT ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT
     3961 TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA
     4021 GTTGGGTGCA CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG
     4081 TTTTCGCCCC GAAGAACGTT TTCCAATGAT GAGCACTTTT AAAGTTCTGC TATGTGGCGC
     4141 GGTATTATCC CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA
     4201 GAATGACTTG GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT
     4261 AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT
     4321 GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT
     4381 AACTCGCCTT GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA
     4441 CACCACGATG CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT
     4501 TACTCTAGCT TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC
     4561 ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA
     4621 GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT
     4681 AGTTATCTAC ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA
     4741 GATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC CAAGTTTACT CATATATACT
     4801 TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA
     4861 TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT CAGACCCCGT
     4921 AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG CGCGTAATCT GCTGCTTGCA
     4981 AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG GATCAAGAGC TACCAACTCT
     5041 TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA AATACTGTCC TTCTAGTGTA
     5101 GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT
     5161 AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG GGTTGGACTC
     5221 AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT CGTGCACACA
     5281 GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC CTACAGCGTG AGCTATGAGA
     5341 AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG
     5401 AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT
     5461 CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG GGGGGCGGAG
     5521 CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC CTGGCCTTTT GCTGGCCTTT
     5581 TGCTCACATG T
//