LOCUS       C-term_AAV-SpCas9-VRQR-gRNA_entry_(HES1477)        8268 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
     terminator      complement(7..13)
                     /created_by="User"
                     /label="U6 terminator"
     ncRNA           complement(14..89)
                     /created_by="User"
                     /label="SpCas9 gRNA scaffold"
     misc_feature    97..991
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /label="RFP dropout cassette"
     misc_feature    complement(97..234)
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /label="Terminator"
     CDS             complement(235..912)
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /Color="#ff1111"
                     /label="RFP"
     misc_feature    complement(913..991)
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /label="Tet Promoter"
     promoter        complement(1006..1262)
                     /created_by="User"
                     /label="hU6"
     repeat_region   complement(<1295..1400)
                     /vntifkey=34
                     /modified_by="jonlevy"
                     /label="3'-ITR pSub201(+)"
     reporter        1416..1576
                     /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    1594..1900
                     /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        2093..2121
                     /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          2163..3023
                     /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    3178..3797
                     /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        4106..4135
                     /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   4211..4340
                     /vntifkey=34
                     /modified_by="jonlevy"
                     /label="5'-ITR pSub201(+)"
     promoter        4357..5161
                     /label="CBh promoter"
     promoter        4365..4650
                     /NC_006273.2="175569-175266"
                     /created_by="User"
                     /modified_by="User"
                     /label="CMV Enhancer"
     promoter        4663..4924
                     /created_by="User"
                     /X00182.1="280-540, +2"
                     /modified_by="User"
                     /label="Chicken B-actin promoter"
     promoter        4901..4906
                     /created_by="User"
                     /label="CBA TATA Box"
     promoter        4925..5064
                     /X00182.1="541-677"
                     /created_by="User"
                     /label="Chicken B-actin intron"
     promoter        4931
                     /created_by="User"
                     /label="putative cba +1"
     promoter        5019..5027
                     /created_by="User"
                     /modified_by="User"
                     /label="b-actin SD (MaxEnt)"
     promoter        5070..5126
                     /NC_001510.1="2312-2368"
                     /created_by="User"
                     /modified_by="User"
                     /label="MVM Intron"
     promoter        5072..5080
                     /created_by="User"
                     /label="MVM SD"
     promoter        5127..5161
                     /From="methods of http://dx.doi.org/10.1038/sj.mt.6300355"
                     /created_by="User"
                     /label="MVM Intron 2"
     promoter        complement(5154..5158)
                     /created_by="User"
                     /label="MVM SA (MaxEnt)"
     motif           5168..5177
                     /created_by="benjaminkleinstiver"
                     /label="Kozak"
     CDS             5174..5230
                     /created_by="jonlevy"
                     /Transferred_From="N-terminal BPNLS"
                     /Transferred_Similarity="100.00%"
                     /Color="#6bba70"
                     /label="BPNLS"
     CDS             5231..5335
                     /created_by="jonlevy"
                     /modified_by="jonlevy"
                     /Color="#0077ee"
                     /label="NpuC"
     misc_marker     5336..5338
                     /created_by="ben"
                     /label="SpCas9 C574"
     CDS             <5336..7720
                     /codon_start=1
                     /note="hSpCas9"
                     /modified_by="jonlevy"
                     /modified_by="benjaminkleinstiver"
                     /label="SpCas9-VRQR (C-term)"
     misc_difference 7019..7021
                     /created_by="bpk6"
                     /modified_by="ben"
                     /label="D1135V"
     misc_difference 7268..7270
                     /created_by="bpk6"
                     /modified_by="ben"
                     /label="G1218R"
     misc_difference 7619..7621
                     /created_by="bpk6"
                     /modified_by="ben"
                     /label="R1335Q"
     misc_difference 7625..7627
                     /created_by="bpk6"
                     /modified_by="ben"
                     /label="T1337R"
     CDS             7721..7783
                     /created_by="jonlevy"
                     /Color="#6bba70"
                     /label="BPNLS"
     CDS             7784..7786
                     /Color="#000000"
                     /created_by="benjaminkleinstiver"
                     /label
     terminator      7792..7952
                     /created_by="User"
                     /modified_by="User"
                     /label="WPRE_Gamma"
     Terminator      8041..8268
                     /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"
ORIGIN      
        1 ctcgagaaaa aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta
       61 ttttaacttg ctatttctag ctctaaaacc gagacgtata aacgcagaaa ggcccacccg
      121 aaggtgagcc agtgtgactc tagtagagag cgttcaccga caaacaacag ataaaacgaa
      181 aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggagatc cttattaagc
      241 acccgtggag tgacgacctt cagcacgttc gtactgttca acgatggtgt agtcttcgtt
      301 gtgggaggtg atgtccagtt tgatgtcggt tttgtaagca cccggcagct gaacgggttt
      361 tttagccatg taggtggttt taacttcagc gtcgtagtga ccaccgtctt tcagtttcag
      421 acgcattttg atttcacctt tcagagcacc gtcttccggg tacatacgtt cggtggaagc
      481 ttcccaaccc atggtttttt tctgcataac cggaccgtcg gacgggaagt tggtaccacg
      541 cagtttaact ttgtagatga actcaccgtc ttgcagggag gagtcctggg taacggtaac
      601 aacaccaccg tcttcgaagt tcataacacg ttcccatttg aaaccttccg ggaaggacag
      661 tttcaggtag tccgggatgt cagccgggtg tttaacgtaa gctttggaac cgtactggaa
      721 ctgcggggac aggatgtccc aagcgaacgg cagcggacca cctttggtaa ctttcagttt
      781 agcggtctgg gtaccttcgt acggacgacc ttcaccttca ccttcgattt cgaactcgtg
      841 accgttaacg gaaccttcca tacgaacttt gaaacgcatg aactctttga taacgtcttc
      901 gctactcgcc atagatcctt tctcctcttt cagatccgtg ctcagtatct ctatcactga
      961 tagggatgtc aatctctatc actgataggg acgtctcggg tgtttcgtcc tttccacaag
     1021 atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg atagtccatt
     1081 ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct acgtcacgta
     1141 ttttgtacta atatctttgt gtttacagtc aaattaattc taattatctc tctaacagcc
     1201 ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctcttc ctgcccgacc
     1261 ttgcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     1321 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     1381 gtgagcgagc gagcgcgcag ccttaattaa cctaattcac tggccgtcgt tttacaacgt
     1441 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     1501 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     1561 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1621 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     1681 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     1741 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     1801 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     1861 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     1921 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     1981 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     2041 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2101 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2161 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     2221 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     2281 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     2341 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     2401 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     2461 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     2521 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     2581 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     2641 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     2701 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     2761 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     2821 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     2881 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     2941 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     3001 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3061 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3121 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3181 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3241 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3301 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     3361 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3421 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3481 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3541 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3601 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3661 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3721 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3781 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3841 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3901 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3961 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     4021 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     4081 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     4141 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccagat
     4201 ttaattaagg ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg
     4261 ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa
     4321 ctccatcact aggggttcct gcggcctcta gatcagggta cccgttacat aacttacggt
     4381 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc
     4441 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     4501 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     4561 gcccgcctgg cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     4621 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     4681 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
     4741 tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga
     4801 ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg
     4861 aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg
     4921 gcgggcggga gtcgctgcga cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc
     4981 gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc
     5041 cttctcctcc gggctgtaat tagctgagca agaggtaagg gtttaaggga tggttggttg
     5101 gtggggtatt aatgtttaat tacctggagc acctgcctga aatcactttt tttcaggttg
     5161 gaccggtgcc accatgaaac ggacagccga cggaagcgag ttcgagtcac caaagaagaa
     5221 gcggaaagtc atcaagattg ctacacggaa atacctggga aagcagaacg tgtacgacat
     5281 cggcgtggag cgggatcaca acttcgccct gaagaatggc tttatcgcca gcaattgctt
     5341 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca
     5401 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat
     5461 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg
     5521 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag
     5581 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc
     5641 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca
     5701 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg
     5761 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
     5821 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
     5881 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
     5941 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
     6001 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
     6061 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
     6121 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa
     6181 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt
     6241 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
     6301 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
     6361 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
     6421 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
     6481 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
     6541 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac
     6601 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt
     6661 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa
     6721 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
     6781 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
     6841 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
     6901 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
     6961 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcgt
     7021 gagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
     7081 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
     7141 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
     7201 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
     7261 ctctgccaga gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
     7321 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa
     7381 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga
     7441 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa
     7501 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct
     7561 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagca
     7621 gtacagaagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct
     7681 gtacgagaca cggatcgacc tgtctcagct gggaggtgac tctggcggct caaaaagaac
     7741 cgccgacggc agcgaattcg agcccaagaa gaagaggaaa gtctaagatc tgataatcaa
     7801 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     7861 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     7921 ttcattttct cctccttgta taaatcctgg ttagttcttg ccacggcgga actcatcgcc
     7981 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     8041 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     8101 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     8161 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     8221 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgg
//
