LOCUS       PX552-CMV-TD-V1         5592 bp ds-DNA     circular     05-OCT-2023
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    406..425
                     /label="px552-Tandem V1 site (mu/hu/mk)"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     misc_RNA        427..502
                     /label="gRNA scaffold"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     primer          complement(497..521)
                     /label="px552_insert_RVS with PspOMI"
                     /note="sequence: TCTGCAGTCTAGAAAAAAAGCACCG"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     terminator      503..508
                     /label="pol III terminator"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     misc_feature    518..522
                     /label="PspOMI RE Site"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     primer          complement(544..571)
                     /label="Seq-px552 tandem-RVS"
                     /note="sequence: CGCCTAACCCTAAGCAGATTCTTCATGC"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
     primer          complement(587..609)
                     /label="Seq-px552 tandem-RVS2"
                     /note="sequence: GCGTATATCTGGCCCGTACATCG"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     enhancer        613..992
                     /label="CMV enhancer"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     primer          complement(651..673)
                     /label="Seq-px552 tandem-RVS3"
                     /note="sequence: GGGCTATGAACTAATGACCCCGT"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     promoter        994..1192
                     /label="CMV promoter"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     misc_feature    complement(1287..1294)
                     /label="BamHI RE Site"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
     CDS             1306..1308
                     /label="EGFP"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     CDS             1309..1311
                     /label="EGFP"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     CDS             1312..2022
                     /label="EGFP"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     primer          1805..1825
                     /label="eGFP-F2-qPCR-SYBRG"
                     /note="sequence: agatccgccacaacatcgagg"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     primer          complement(1952..1970)
                     /label="eGFP-R2-qPCR-SYBRG"
                     /note="sequence: agcaggaccatgtgatcgc"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     CDS             2041..2289
                     /label="KASH"
                     /ApEinfo_revcolor="#993366"
                     /ApEinfo_fwdcolor="#993366"
     polyA_signal    2339..2815
                     /label="hGH poly(A) signal"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     repeat_region   2855..2995
                     /label="AAV2 ITR"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
     rep_origin      3070..3525
                     /label="f1 ori"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     promoter        3807..3911
                     /label="AmpR promoter"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     CDS             3912..3980
                     /label="AmpR"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     CDS             3981..4772
                     /label="AmpR"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     rep_origin      4943..5531
                     /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 CACCGCAGAA AGTTCATGGT
      421 TTCGGTGTTT TAGAGCTAGA AATAGCAAGT TAAAATAAGG CTAGTCCGTT ATCAACTTGA
      481 AAAAGTGGCA CCGAGTCGGT GCTTTTTTTC TAGACTGCAG AGGGCCCGGC TTGACCGACA
      541 ATTGCATGAA GAATCTGCTT AGGGTTAGGC GTTTTGCGCT GCTTCGCGAT GTACGGGCCA
      601 GATATACGCG TTGACATTGA TTATTGACTA GTTATTAATA GTAATCAATT ACGGGGTCAT
      661 TAGTTCATAG CCCATATATG GAGTTCCGCG TTACATAACT TACGGTAAAT GGCCCGCCTG
      721 GCTGACCGCC CAACGACCCC CGCCCATTGA CGTCAATAAT GACGTATGTT CCCATAGTAA
      781 CGCCAATAGG GACTTTCCAT TGACGTCAAT GGGTGGAGTA TTTACGGTAA ACTGCCCACT
      841 TGGCAGTACA TCAAGTGTAT CATATGCCAA GTACGCCCCC TATTGACGTC AATGACGGTA
      901 AATGGCCCGC CTGGCATTAT GCCCAGTACA TGACCTTATG GGACTTTCCT ACTTGGCAGT
      961 ACATCTACGT ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG TACATCAATG
     1021 GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG
     1081 GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC
     1141 CATTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGCGCGTTTT
     1201 GCCTGTACTG GGTCTCTCTG GTTAGACCAG ATCTGAGCCT GGGAGCTCTC TGGCTAACTA
     1261 GGGAACCCAC TGCTTAAGCC GGATCCCTCT AGAGTCGACG CCACCATGGT GAGCAAGGGC
     1321 GAGGAGCTGT TCACCGGGGT GGTGCCCATC CTGGTCGAGC TGGACGGCGA CGTAAACGGC
     1381 CACAAGTTCA GCGTGTCCGG CGAGGGCGAG GGCGATGCCA CCTACGGCAA GCTGACCCTG
     1441 AAGTTCATCT GCACCACCGG CAAGCTGCCC GTGCCCTGGC CCACCCTCGT GACCACCCTG
     1501 ACCTACGGCG TGCAGTGCTT CAGCCGCTAC CCCGACCACA TGAAGCAGCA CGACTTCTTC
     1561 AAGTCCGCCA TGCCCGAAGG CTACGTCCAG GAGCGCACCA TCTTCTTCAA GGACGACGGC
     1621 AACTACAAGA CCCGCGCCGA GGTGAAGTTC GAGGGCGACA CCCTGGTGAA CCGCATCGAG
     1681 CTGAAGGGCA TCGACTTCAA GGAGGACGGC AACATCCTGG GGCACAAGCT GGAGTACAAC
     1741 TACAACAGCC ACAACGTCTA TATCATGGCC GACAAGCAGA AGAACGGCAT CAAGGTGAAC
     1801 TTCAAGATCC GCCACAACAT CGAGGACGGC AGCGTGCAGC TCGCCGACCA CTACCAGCAG
     1861 AACACCCCCA TCGGCGACGG CCCCGTGCTG CTGCCCGACA ACCACTACCT GAGCACCCAG
     1921 TCCGCCCTGA GCAAAGACCC CAACGAGAAG CGCGATCACA TGGTCCTGCT GGAGTTCGTG
     1981 ACCGCCGCCG GGATCACTCT CGGCATGGAC GAGCTGTACA AGTCCGGACT CAGATCTCGA
     2041 GAGGAGGAGG AGGAGACAGA CAGCAGGATG CCCCACCTCG ACAGCCCCGG CAGCTCCCAG
     2101 CCGAGACGCT CCTTCCTCTC AAGGGTGATC AGGGCAGCGC TACCGTTGCA GCTGCTTCTG
     2161 CTGCTGCTGC TGCTCCTGGC CTGCCTGCTA CCTGCCTCTG AAGATGACTA CAGCTGCACC
     2221 CAGGCCAACA ACTTTGCCCG ATCCTTCTAC CCCATGCTGC GGTACACCAA CGGGCCACCT
     2281 CCCACCTAGG AATTCGATAT CAAGCTTATC GATACCGAGC GCTGCTCGAG AGATCTACGG
     2341 GTGGCATCCC TGTGACCCCT CCCCAGTGCC TCTCCTGGCC CTGGAAGTTG CCACTCCAGT
     2401 GCCCACCAGC CTTGTCCTAA TAAAATTAAG TTGCATCATT TTGTCTGACT AGGTGTCCTT
     2461 CTATAATATT ATGGGGTGGA GGGGGGTGGT ATGGAGCAAG GGGCAAGTTG GGAAGACAAC
     2521 CTGTAGGGCC TGCGGGGTCT ATTGGGAACC AAGCTGGAGT GCAGTGGCAC AATCTTGGCT
     2581 CACTGCAATC TCCGCCTCCT GGGTTCAAGC GATTCTCCTG CCTCAGCCTC CCGAGTTGTT
     2641 GGGATTCCAG GCATGCATGA CCAGGCTCAG CTAATTTTTG TTTTTTTGGT AGAGACGGGG
     2701 TTTCACCATA TTGGCCAGGC TGGTCTCCAA CTCCTAATCT CAGGTGATCT ACCCACCTTG
     2761 GCCTCCCAAA TTGCTGGGAT TACAGGCGTG AACCACTGCT CCCTTCCCTG TCCTTCTGAT
     2821 TTTGTAGGTA ACCACGTGCG GACCGAGCGG CCGCAGGAAC CCCTAGTGAT GGAGTTGGCC
     2881 ACTCCCTCTC TGCGCGCTCG CTCGCTCACT GAGGCCGGGC GACCAAAGGT CGCCCGACGC
     2941 CCGGGCTTTG CCCGGGCGGC CTCAGTGAGC GAGCGAGCGC GCAGCTGCCT GCAGGGGCGC
     3001 CTGATGCGGT ATTTTCTCCT TACGCATCTG TGCGGTATTT CACACCGCAT ACGTCAAAGC
     3061 AACCATAGTA CGCGCCCTGT AGCGGCGCAT TAAGCGCGGC GGGTGTGGTG GTTACGCGCA
     3121 GCGTGACCGC TACACTTGCC AGCGCCCTAG CGCCCGCTCC TTTCGCTTTC TTCCCTTCCT
     3181 TTCTCGCCAC GTTCGCCGGC TTTCCCCGTC AAGCTCTAAA TCGGGGGCTC CCTTTAGGGT
     3241 TCCGATTTAG TGCTTTACGG CACCTCGACC CCAAAAAACT TGATTTGGGT GATGGTTCAC
     3301 GTAGTGGGCC ATCGCCCTGA TAGACGGTTT TTCGCCCTTT GACGTTGGAG TCCACGTTCT
     3361 TTAATAGTGG ACTCTTGTTC CAAACTGGAA CAACACTCAA CCCTATCTCG GGCTATTCTT
     3421 TTGATTTATA AGGGATTTTG CCGATTTCGG CCTATTGGTT AAAAAATGAG CTGATTTAAC
     3481 AAAAATTTAA CGCGAATTTT AACAAAATAT TAACGTTTAC AATTTTATGG TGCACTCTCA
     3541 GTACAATCTG CTCTGATGCC GCATAGTTAA GCCAGCCCCG ACACCCGCCA ACACCCGCTG
     3601 ACGCGCCCTG ACGGGCTTGT CTGCTCCCGG CATCCGCTTA CAGACAAGCT GTGACCGTCT
     3661 CCGGGAGCTG CATGTGTCAG AGGTTTTCAC CGTCATCACC GAAACGCGCG AGACGAAAGG
     3721 GCCTCGTGAT ACGCCTATTT TTATAGGTTA ATGTCATGAT AATAATGGTT TCTTAGACGT
     3781 CAGGTGGCAC TTTTCGGGGA AATGTGCGCG GAACCCCTAT TTGTTTATTT TTCTAAATAC
     3841 ATTCAAATAT GTATCCGCTC ATGAGACAAT AACCCTGATA AATGCTTCAA TAATATTGAA
     3901 AAAGGAAGAG TATGAGTATT CAACATTTCC GTGTCGCCCT TATTCCCTTT TTTGCGGCAT
     3961 TTTGCCTTCC TGTTTTTGCT CACCCAGAAA CGCTGGTGAA AGTAAAAGAT GCTGAAGATC
     4021 AGTTGGGTGC ACGAGTGGGT TACATCGAAC TGGATCTCAA CAGCGGTAAG ATCCTTGAGA
     4081 GTTTTCGCCC CGAAGAACGT TTTCCAATGA TGAGCACTTT TAAAGTTCTG CTATGTGGCG
     4141 CGGTATTATC CCGTATTGAC GCCGGGCAAG AGCAACTCGG TCGCCGCATA CACTATTCTC
     4201 AGAATGACTT GGTTGAGTAC TCACCAGTCA CAGAAAAGCA TCTTACGGAT GGCATGACAG
     4261 TAAGAGAATT ATGCAGTGCT GCCATAACCA TGAGTGATAA CACTGCGGCC AACTTACTTC
     4321 TGACAACGAT CGGAGGACCG AAGGAGCTAA CCGCTTTTTT GCACAACATG GGGGATCATG
     4381 TAACTCGCCT TGATCGTTGG GAACCGGAGC TGAATGAAGC CATACCAAAC GACGAGCGTG
     4441 ACACCACGAT GCCTGTAGCA ATGGCAACAA CGTTGCGCAA ACTATTAACT GGCGAACTAC
     4501 TTACTCTAGC TTCCCGGCAA CAATTAATAG ACTGGATGGA GGCGGATAAA GTTGCAGGAC
     4561 CACTTCTGCG CTCGGCCCTT CCGGCTGGCT GGTTTATTGC TGATAAATCT GGAGCCGGTG
     4621 AGCGTGGGTC TCGCGGTATC ATTGCAGCAC TGGGGCCAGA TGGTAAGCCC TCCCGTATCG
     4681 TAGTTATCTA CACGACGGGG AGTCAGGCAA CTATGGATGA ACGAAATAGA CAGATCGCTG
     4741 AGATAGGTGC CTCACTGATT AAGCATTGGT AACTGTCAGA CCAAGTTTAC TCATATATAC
     4801 TTTAGATTGA TTTAAAACTT CATTTTTAAT TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG
     4861 ATAATCTCAT GACCAAAATC CCTTAACGTG AGTTTTCGTT CCACTGAGCG TCAGACCCCG
     4921 TAGAAAAGAT CAAAGGATCT TCTTGAGATC CTTTTTTTCT GCGCGTAATC TGCTGCTTGC
     4981 AAACAAAAAA ACCACCGCTA CCAGCGGTGG TTTGTTTGCC GGATCAAGAG CTACCAACTC
     5041 TTTTTCCGAA GGTAACTGGC TTCAGCAGAG CGCAGATACC AAATACTGTC CTTCTAGTGT
     5101 AGCCGTAGTT AGGCCACCAC TTCAAGAACT CTGTAGCACC GCCTACATAC CTCGCTCTGC
     5161 TAATCCTGTT ACCAGTGGCT GCTGCCAGTG GCGATAAGTC GTGTCTTACC GGGTTGGACT
     5221 CAAGACGATA GTTACCGGAT AAGGCGCAGC GGTCGGGCTG AACGGGGGGT TCGTGCACAC
     5281 AGCCCAGCTT GGAGCGAACG ACCTACACCG AACTGAGATA CCTACAGCGT GAGCTATGAG
     5341 AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG CGGACAGGTA TCCGGTAAGC GGCAGGGTCG
     5401 GAACAGGAGA GCGCACGAGG GAGCTTCCAG GGGGAAACGC CTGGTATCTT TATAGTCCTG
     5461 TCGGGTTTCG CCACCTCTGA CTTGAGCGTC GATTTTTGTG ATGCTCGTCA GGGGGGCGGA
     5521 GCCTATGGAA AAACGCCAGC AACGCGGCCT TTTTACGGTT CCTGGCCTTT TGCTGGCCTT
     5581 TTGCTCACAT GT
//