LOCUS       C-term_AAV-eVRQR-ACTA2-gRNA-A4_(mouse)_(LLH310)        7380 bp    DNA     circular UNA 05-OCT-2025
DEFINITION  derivative of JL646-Cbh_v5-C-term_AAV.
ACCESSION
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
FEATURES             Location/Qualifiers
     repeat_region   complement(<1..106)
                     /vntifkey=34
                     /modified_by="jonlevy"
                     /label="3'-ITR pSub201(+)"
     reporter        122..282
                     /Imported_from="<a
                     href=""http://wishart.biology.ualberta.ca/PlasMapper"">Pla
                     sMapper</a>"
                     /Transferred_From="lacZ_a reporter"
                     /Transferred_Similarity="100.00%"
                     /label="lacZ_a reporter"
     misc_feature    300..606
                     /Imported_from="<a
                     href=""http://wishart.biology.ualberta.ca/PlasMapper"">Pla
                     sMapper</a>"
                     /Transferred_From="f1 origin"
                     /Transferred_Similarity="99.67%"
                     /label="f1 origin"
                     /note="Geneious type: origin of replication"
     promoter        799..827
                     /Imported_from="<a
                     href=""http://wishart.biology.ualberta.ca/PlasMapper"">Pla
                     sMapper</a>"
                     /Transferred_From="amp prom"
                     /Transferred_Similarity="100.00%"
                     /label="amp prom"
     marker          869..1729
                     /Imported_from="<a
                     href=""http://wishart.biology.ualberta.ca/PlasMapper"">Pla
                     sMapper</a>"
                     /Transferred_From="amp marker"
                     /Transferred_Similarity="99.42%"
                     /label="amp marker"
     misc_feature    1884..2503
                     /Imported_from="<a
                     href=""http://wishart.biology.ualberta.ca/PlasMapper"">Pla
                     sMapper</a>"
                     /Transferred_From="pBR322 origin"
                     /Transferred_Similarity="99.68%"
                     /label="pBR322 origin"
                     /note="Geneious type: origin of replication"
     promoter        2812..2841
                     /Imported_from="<a
                     href=""http://wishart.biology.ualberta.ca/PlasMapper"">Pla
                     sMapper</a>"
                     /Transferred_From="lac prom"
                     /Transferred_Similarity="100.00%"
                     /label="lac prom"
     repeat_region   2917..3046
                     /vntifkey=34
                     /modified_by="jonlevy"
                     /label="5'-ITR pSub201(+)"
     promoter        3063..3867
                     /label="CBh promoter"
     promoter        3071..3356
                     /NC_006273.2="175569-175266"
                     /created_by="User"
                     /modified_by="User"
                     /label="CMV Enhancer"
     promoter        3369..3630
                     /created_by="User"
                     /X00182.1="280-540, +2"
                     /modified_by="User"
                     /label="Chicken B-actin promoter"
     promoter        3607..3612
                     /created_by="User"
                     /label="CBA TATA Box"
     promoter        3631..3770
                     /X00182.1="541-677"
                     /created_by="User"
                     /label="Chicken B-actin intron"
     promoter        3637
                     /created_by="User"
                     /label="putative cba +1"
     promoter        3725..3733
                     /created_by="User"
                     /modified_by="User"
                     /label="b-actin SD (MaxEnt)"
     promoter        3776..3832
                     /NC_001510.1="2312-2368"
                     /created_by="User"
                     /modified_by="User"
                     /label="MVM Intron"
     promoter        3778..3786
                     /created_by="User"
                     /label="MVM SD"
     promoter        3833..3867
                     /From="methods of http://dx.doi.org/10.1038/sj.mt.6300355"
                     /created_by="User"
                     /label="MVM Intron 2"
     promoter        complement(3860..3864)
                     /created_by="User"
                     /label="MVM SA (MaxEnt)"
     motif           3874..3883
                     /created_by="benjaminkleinstiver"
                     /label="Kozak"
     CDS             3880..3936
                     /created_by="jonlevy"
                     /Transferred_From="N-terminal BPNLS"
                     /Transferred_Similarity="100.00%"
                     /Color="#6bba70"
                     /label="BPNLS"
     CDS             3937..4041
                     /created_by="jonlevy"
                     /modified_by="jonlevy"
                     /Color="#0077ee"
                     /label="NpuC"
     misc_marker     4042..4044
                     /created_by="ben"
                     /label="SpCas9 C574"
     CDS             <4042..6426
                     /codon_start=1
                     /note="hSpCas9"
                     /modified_by="jonlevy"
                     /modified_by="benjaminkleinstiver"
                     /label="SpCas9-VRQR (C-term)"
     misc_difference 5725..5727
                     /created_by="bpk6"
                     /modified_by="ben"
                     /label="D1135V"
     misc_difference 5974..5976
                     /created_by="bpk6"
                     /modified_by="ben"
                     /label="G1218R"
     misc_difference 6325..6327
                     /created_by="bpk6"
                     /modified_by="ben"
                     /label="R1335Q"
     misc_difference 6331..6333
                     /created_by="bpk6"
                     /modified_by="ben"
                     /label="T1337R"
     CDS             6427..6489
                     /created_by="jonlevy"
                     /Color="#6bba70"
                     /label="BPNLS"
     CDS             6490..6492
                     /Color="#000000"
                     /created_by="benjaminkleinstiver"
                     /label
     terminator      6498..6658
                     /created_by="User"
                     /modified_by="User"
                     /label="WPRE_Gamma"
     Terminator      6747..6974
                     /Imported_from="<a
                     href=""http://wishart.biology.ualberta.ca/PlasMapper"">Pla
                     sMapper</a>"
                     /Transferred_From="bGH_PA term"
                     /Transferred_Similarity="100.00%"
                     /label="bGH_PA term"
     terminator      complement(6981..6987)
                     /created_by="User"
                     /label="U6 terminator"
     misc_RNA        complement(6988..7063)
                     /created_by="User"
                     /modified_by="benk"
                     /label="SpCas9 gRNA scaffold"
     misc_marker     complement(7064..7083)
                     /created_by="benk"
                     /modified_by="ben"
                     /label="ACTA2 R179H A4 mouse gRNA spacer"
     promoter        complement(7092..7348)
                     /created_by="User"
                     /label="hU6"
ORIGIN      
        1 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
       61 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagcctt aattaaccta
      121 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
      181 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
      241 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
      301 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
      361 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
      421 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
      481 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
      541 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
      601 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
      661 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
      721 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      781 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      841 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      901 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      961 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1021 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1081 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     1141 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1201 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1261 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1321 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1381 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1441 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1501 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1561 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1621 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1681 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1741 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1801 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1861 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1921 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1981 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2041 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2101 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2161 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2221 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2281 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2341 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2401 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2461 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2521 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2581 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2641 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     2701 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     2761 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     2821 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     2881 cagctatgac catgattacg ccagatttaa ttaaggctgc gcgctcgctc gctcactgag
     2941 gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     3001 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagatc
     3061 agggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     3121 cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca atgggtggag
     3181 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     3241 cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta catgacctta
     3301 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag
     3361 gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg
     3421 tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc
     3481 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg
     3541 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     3601 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc
     3661 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
     3721 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc tgagcaagag
     3781 gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc tggagcacct
     3841 gcctgaaatc actttttttc aggttggacc ggtgccacca tgaaacggac agccgacgga
     3901 agcgagttcg agtcaccaaa gaagaagcgg aaagtcatca agattgctac acggaaatac
     3961 ctgggaaagc agaacgtgta cgacatcggc gtggagcggg atcacaactt cgccctgaag
     4021 aatggcttta tcgccagcaa ttgcttcgac tccgtggaaa tctccggcgt ggaagatcgg
     4081 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc
     4141 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt
     4201 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa
     4261 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg
     4321 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac
     4381 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag
     4441 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat
     4501 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag
     4561 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag
     4621 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag
     4681 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg
     4741 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag
     4801 gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt
     4861 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag
     4921 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg
     4981 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc
     5041 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag
     5101 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat
     5161 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc
     5221 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac
     5281 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc
     5341 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag
     5401 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc
     5461 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac
     5521 ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg
     5581 ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc
     5641 aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg
     5701 gaccctaaga agtacggcgg cttcgtgagc cccaccgtgg cctattctgt gctggtggtg
     5761 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc
     5821 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc
     5881 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg
     5941 gaaaacggcc ggaagagaat gctggcctct gccagagaac tgcagaaggg aaacgaactg
     6001 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag
     6061 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg
     6121 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat
     6181 ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc
     6241 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac
     6301 tttgacacca ccatcgaccg gaagcagtac agaagcacca aagaggtgct ggacgccacc
     6361 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga
     6421 ggtgactctg gcggctcaaa aagaaccgcc gacggcagcg aattcgagcc caagaagaag
     6481 aggaaagtct aagatctgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     6541 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     6601 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttag
     6661 ttcttgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     6721 tgttgggcac tgacaattcc gtggtgcgac tgtgccttct agttgccagc catctgttgt
     6781 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     6841 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     6901 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     6961 ggtgggctct atggctcgag aaaaaaagca ccgactcggt gccacttttt caagttgata
     7021 acggactagc cttattttaa cttgctattt ctagctctaa aacgccagcc aagtccagat
     7081 gcacggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa tactttcaag
     7141 ttacggtaag catatgatag tccattttaa aacataattt taaaactgca aactacccaa
     7201 gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat
     7261 taattctaat tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg
     7321 gaaataggcc ctcttcctgc ccgaccttgc ggccgcagga acccctagtg atggagttgg
//
