LOCUS       PEA1-nuclease          11070 bp ds-DNA     circular     02-SEP-2021
DEFINITION  .
FEATURES             Location/Qualifiers
     promoter        1..241
                     /label="U6 promoter"
     misc_feature    246..267
                     /label="Gate gRNA 1"
     misc_RNA        268..343
                     /label="gRNA scaffold"
     misc_feature    344..365
                     /label="Gate RT template"
     enhancer        428..713
                     /label="CMV enhancer"
     misc_feature    714..988
                     /label="Chicken B-actin promoter"
     intron          989..1216
                     /label="hybrid intron"
     regulatory      complement(1228..1237)
                     /label="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)"
     CDS             1234..7655
                     /label="Translation 1234-7655"
     CDS             1237..1302
                     /label="3xFLAG"
     CDS             1309..1329
                     /label="SV40 NLS"
     misc_feature    1354..5454
                     /label="Cas9"
     misc_feature    5367..5367
                     /label="originally C, replaced with G to remove Xcm1"
     misc_feature    5455..7626
                     /label="reverse transcriptase"
     misc_feature    5922..5922
                     /label="originally A, replaced with G to remove Bbs1"
     misc_feature    6435..6435
                     /label="originally G, replaced with A to remove Bbs1"
     misc_feature    6603..6603
                     /label="originally T, replaced with C to remove Sap1"
     misc_feature    7627..7655
                     /label="nucleoplasmin NLS"
     primer_bind     complement(7702..7719)
                     /label="BGH-rev"
     polyA_signal    7708..7915
                     /label="bGH poly(A) signal"
     primer_bind     7924..7940
                     /label="pBluescriptSK"
     primer_bind     7924..7940
                     /label="SK primer"
     misc_RNA        complement(7983..8058)
                     /label="gRNA scaffold"
     misc_RNA        complement(8059..8078)
                     /label="Gate gRNA 2"
     promoter        complement(8084..8324)
                     /label="U6 promoter"
     repeat_region   8333..8462
                     /label="AAV2 ITR"
     rep_origin      8548..9003
                     /label="f1 ori"
     primer_bind     complement(8635..8654)
                     /label="F1ori-R"
     primer_bind     8845..8866
                     /label="F1ori-F"
     primer_bind     complement(9020..9039)
                     /label="pRS-marker"
     primer_bind     9139..9161
                     /label="pGEX 3'"
     primer_bind     complement(9199..9217)
                     /label="pBRforEco"
     promoter        9285..9389
                     /label="AmpR promoter"
     primer_bind     complement(9608..9627)
                     /label="Amp-R"
     CDS             9809..10250
                     /label="AmpR"
     rep_origin      10421..11009
                     /label="ori"
     primer_bind     10910..10929
                     /label="pBR322ori-F"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcgggtctt cgagaagacc
      361 ttttttttgt tctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa atggctctag
      421 aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      481 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      541 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      601 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
      661 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
      721 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
      781 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggcgcgcgcc
      841 aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcagc
      901 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc
      961 ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc
     1021 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
     1081 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg
     1141 gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga
     1201 aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca cgacggagac
     1261 tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc aaagaagaag
     1321 cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat cggcctggac
     1381 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag
     1441 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc
     1501 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga
     1561 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg
     1621 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat
     1681 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag
     1741 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac
     1801 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc
     1861 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag
     1921 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc
     1981 atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc
     2041 ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc
     2101 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc
     2161 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt
     2221 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc
     2281 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag
     2341 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt
     2401 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa
     2461 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc
     2521 gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc
     2581 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac
     2641 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac
     2701 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag
     2761 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc
     2821 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag
     2881 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg
     2941 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac
     3001 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag
     3061 aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc
     3121 ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag
     3181 gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag
     3241 atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag
     3301 ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc
     3361 cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac
     3421 agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa
     3481 gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc
     3541 cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg
     3601 atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc
     3661 cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag
     3721 ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag
     3781 ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc
     3841 aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac
     3901 tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg
     3961 ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag
     4021 ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa
     4081 ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac
     4141 gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc
     4201 cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc
     4261 cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac
     4321 gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac
     4381 ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc
     4441 aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt
     4501 accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg
     4561 gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc
     4621 caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc
     4681 ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag
     4741 tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa
     4801 aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa
     4861 agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg
     4921 aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg
     4981 aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc
     5041 aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag
     5101 gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc
     5161 gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
     5221 ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc
     5281 cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc
     5341 atcgaccgga agaggtacac cagcacgaaa gaggtgctgg acgccaccct gatccaccag
     5401 agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctgga
     5461 ggatctagcg gaggatcctc tggcagcgag acaccaggaa caagcgagtc agcaacacca
     5521 gagagcagtg gcggcagcag cggcggcagc agcaccctaa atatagaaga tgagtatcgg
     5581 ctacatgaga cctcaaaaga gccagatgtt tctctagggt ccacatggct gtctgatttt
     5641 cctcaggcct gggcggaaac cgggggcatg ggactggcag ttcgccaagc tcctctgatc
     5701 atacctctga aagcaacctc tacccccgtg tccataaaac aataccccat gtcacaagaa
     5761 gccagactgg ggatcaagcc ccacatacag agactgttgg accagggaat actggtaccc
     5821 tgccagtccc cctggaacac gcccctgcta cccgttaaga aaccagggac taatgattat
     5881 aggcctgtcc aggatctgag agaagtcaac aagcgggtgg aggacatcca ccccaccgtg
     5941 cccaaccctt acaacctctt gagcgggctc ccaccgtccc accagtggta cactgtgctt
     6001 gatttaaagg atgccttttt ctgcctgaga ctccacccca ccagtcagcc tctcttcgcc
     6061 tttgagtgga gagatccaga gatgggaatc tcaggacaat tgacctggac cagactccca
     6121 cagggtttca aaaacagtcc caccctgttt aatgaggcac tgcacagaga cctagcagac
     6181 ttccggatcc agcacccaga cttgatcctg ctacagtacg tggatgactt actgctggcc
     6241 gccacttctg agctagactg ccaacaaggt actcgggccc tgttacaaac cctagggaac
     6301 ctcgggtatc gggcctcggc caagaaagcc caaatttgcc agaaacaggt caagtatctg
     6361 gggtatcttc taaaagaggg tcagagatgg ctgactgagg ccagaaaaga gactgtgatg
     6421 gggcagccta ctccaaagac ccctcgacaa ctaagggagt tcctagggaa ggcaggcttc
     6481 tgtcgcctct tcatccctgg gtttgcagaa atggcagccc ccctgtaccc tctcaccaaa
     6541 ccggggactc tgtttaattg gggcccagac caacaaaagg cctatcaaga aatcaagcaa
     6601 gcccttctaa ctgccccagc cctggggttg ccagatttga ctaagccctt tgaactcttt
     6661 gtcgacgaga agcagggcta cgccaaaggt gtcctaacgc aaaaactggg accttggcgt
     6721 cggccggtgg cctacctgtc caaaaagcta gacccagtag cagctgggtg gcccccttgc
     6781 ctacggatgg tagcagccat tgccgtactg acaaaggatg caggcaagct aaccatggga
     6841 cagccactag tcattctggc cccccatgca gtagaggcac tagtcaaaca accccccgac
     6901 cgctggcttt ccaacgcccg gatgactcac tatcaggcct tgcttttgga cacggaccgg
     6961 gtccagttcg gaccggtggt agccctgaac ccggctacgc tgctcccact gcctgaggaa
     7021 gggctgcaac acaactgcct tgatatcctg gccgaagccc acggaacccg acccgaccta
     7081 acggaccagc cgctcccaga cgccgaccac acctggtaca cggatggaag cagtctctta
     7141 caagagggac agcgtaaggc gggagctgcg gtgaccaccg agaccgaggt aatctgggct
     7201 aaagccctgc cagccgggac atccgctcag cgggctgaac tgatagcact cacccaggcc
     7261 ctaaagatgg cagaaggtaa gaagctaaat gtttatactg atagccgtta tgcttttgct
     7321 actgcccata tccatggaga aatatacaga aggcgtgggt ggctcacatc agaaggcaaa
     7381 gagatcaaaa ataaagacga gatcttggcc ctactaaaag ccctctttct gcccaaaaga
     7441 cttagcataa tccattgtcc aggacatcaa aagggacaca gcgccgaggc tagaggcaac
     7501 cggatggctg accaagcggc ccgaaaggca gccatcacag agactccaga cacctctacc
     7561 ctcctcatag aaaattcatc accctctggc ggctcaaaaa gaaccgccga cggcagcgaa
     7621 ttcgagaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa
     7681 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     7741 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     7801 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     7861 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc
     7921 cgctctagaa ctagtggatc ccccgggctg caggaattcg atacgaatcc tgcaggaaaa
     7981 aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc
     8041 tatttctagc tctaaaacgt cttctcgaag accccggtgt ttcgtccttt ccacaagata
     8101 tataaagcca agaaatcgaa atactttcaa gttacggtaa gcatatgata gtccatttta
     8161 aaacataatt ttaaaactgc aaactaccca agaaattatt actttctacg tcacgtattt
     8221 tgtactaata tctttgtgtt tacagtcaaa ttaattccaa ttatctctct aacagccttg
     8281 tatcgtatat gcaaatatga aggaatcatg ggaaataggc cctcgcggcc gcaggaaccc
     8341 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     8401 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     8461 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     8521 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     8581 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     8641 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     8701 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     8761 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     8821 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     8881 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     8941 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     9001 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     9061 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     9121 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     9181 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     9241 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     9301 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     9361 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     9421 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     9481 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     9541 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     9601 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     9661 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     9721 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     9781 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     9841 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     9901 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     9961 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
    10021 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
    10081 ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg
    10141 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
    10201 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
    10261 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
    10321 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
    10381 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
    10441 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
    10501 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
    10561 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
    10621 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
    10681 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
    10741 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
    10801 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
    10861 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
    10921 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
    10981 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
    11041 tggccttttg ctggcctttt gctcacatgt
//