LOCUS       pAGT8162_(clean_        7053 bp    DNA     circular UNA 20-SEP-2022
DEFINITION  AATG_dNbLbCpf1i(D156R,E925Q)_TTCG.
ACCESSION   urn.local...1589458423872.518 (modified)
VERSION     urn.local...1589458423872.518
KEYWORDS    .
SOURCE      
  ORGANISM  
            .
FEATURES             Location/Qualifiers
     misc_feature    1..4
                     /label="GG-overhang"
     misc_feature    1..4
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    5..2069
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of pAGM3946 (modified) - Fragment 2
                     (concatenated sequence 1)"
     misc_feature    16..17
                     /note="Geneious type: origin"
                     /label="origin"
     CDS             82..1089
                     /label="Spec"
     misc_feature    complement(115..135)
                     /note="Geneious type: primer_bind_reverse"
                     /label="movebrev-ipb"
     misc_feature    1422..2036
                     /label="rep - pMB1"
     misc_feature    1580
                     /note="Geneious type: mutation"
                     /label="C to T"
     primer_bind     1947..1967
                     /label="moveb-ipb"
     rep_origin      2021
                     /label="ORI"
     misc_feature    2070..2073
                     /label="GG-overhang"
     misc_feature    2070..2073
                     /note="Geneious type: ligation"
                     /label="Ligation"
     CDS             order(2071..2470,2601..2957,3049..3257,3366..3636,
                     3738..3989,4111..4443,4568..4894,5031..5752,5874..6326,
                     6463..6822,6944..7051)
                     /label="dttLbCas12a-i"
     CDS             2071..2130
                     /label="LbNLS"
     misc_feature    2074..3048
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of pAGT8159 - Fragment 2 (concatenated
                     sequence 2)"
     misc_feature    2130..2133
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    2130..2466
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_A1_(E1) - Fragment 2
                     (concatenated sequence 1)"
     misc_feature    2134..3048
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of pAGT5880 - Fragment 2 (concatenated
                     sequence 2)"
     misc_feature    2467..2470
                     /note="Geneious type: ligation"
                     /label="Ligation"
     intron          2471..2600
                     /label="I1 (Intron E2f5)"
     misc_feature    2471..2598
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_A2_(I1) - Fragment 2
                     (concatenated sequence 2)"
     misc_feature    2599..2602
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    2603..2958
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_A3_(E2) - Fragment 2
                     (concatenated sequence 3)"
     misc_feature    2726..2728
                     /note="Geneious type: Domain"
                     /label="D156R (low temp tolerant)"
     misc_feature    2726..2728
                     /note="Geneious type: mutation"
                     /label="GAC to AGA"
     intron          2958..3048
                     /label="I2 (Intron Gluc4)"
     misc_feature    2959..2962
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    2963..>3048
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_A4_(I2-UNI) - Fragment 2
                     (concatenated sequence 4)"
     misc_feature    3049..3052
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    3049..3252
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_B1_(E3) - Fragment 2
                     (concatenated sequence 1)"
     misc_feature    3053..3987
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of pAGT5882 - Fragment 2 (concatenated
                     sequence 3)"
     misc_feature    3253..3256
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    3257..3365
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_B2_(I3-UNI) - Fragment 2
                     (concatenated sequence 2)"
     intron          3258..3365
                     /label="I3 (Intron chtog16)"
     misc_feature    3366..3369
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    3370..3637
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_B3_(E4) - Fragment 2
                     (concatenated sequence 3)"
     intron          3637..3737
                     /label="I4 (Intron cdpk3)"
     misc_feature    3638..3641
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    3642..3734
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_B4_(I4-UNI) - Fragment 2
                     (concatenated sequence 4)"
     misc_feature    3735..3738
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    3739..>3987
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_B5_(E5) - Fragment 2
                     (concatenated sequence 5)"
     misc_feature    3988..3991
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    3988..4110
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_C1_(I5-UNI) - Fragment 2
                     (concatenated sequence 1)"
     intron          3990..4110
                     /label="I5 (Intron chtog11)"
     misc_feature    3992..4872
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of pAGT5884 - Fragment 2 (concatenated
                     sequence 4)"
     misc_feature    4111..4114
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    4115..4437
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_C2_(E6) - Fragment 2
                     (concatenated sequence 2)"
     misc_feature    4438..4441
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    4442..4567
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_C3_(I6-UNI) - Fragment 2
                     (concatenated sequence 3)"
     intron          4444..4567
                     /label="I6 (Intron atbg11)"
     misc_feature    4568..4571
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    4572..>4872
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_C4_(E7) - Fragment 2
                     (concatenated sequence 4)"
     misc_feature    4873..4876
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    4873..5712
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of pAGT5886(D925Q_A) - Fragment 2
                     (concatenated sequence 2)"
     misc_feature    4873..5031
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_D1 (I7) - Fragment 2
                     (concatenated sequence 1)"
     misc_feature    4877..5866
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of pAGT6054 - Fragment 2 (concatenated
                     sequence 5)"
     intron          4895..5030
                     /label="I7 (Intron Atatp2)"
     misc_feature    5032..5035
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    5036..>5712
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_D2_(E8) - Fragment 2
                     (concatenated sequence 2)"
     misc_feature    5713..5716
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    <5713..5751
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_D2_(E8) - Fragment 2
                     (concatenated sequence 2)"
     misc_feature    5714..5716
                     /note="Geneious type: Catalytic"
                     /note="Geneious type: mutation"
                     /label="E925Q"
     misc_feature    5717..>5866
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of pAGT5886(D925Q_B) - Fragment 2
                     (concatenated sequence 3)"
     misc_feature    5752..5755
                     /note="Geneious type: ligation"
                     /label="Ligation"
     intron          5753..5873
                     /label="I8 (Intron Rlk7)"
     misc_feature    5756..>5866
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_D3_(I8-UNI) - Fragment 2
                     (concatenated sequence 3)"
     misc_feature    5867..5870
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    5867..6324
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_E1_(E9) - Fragment 2
                     (concatenated sequence 1)"
     misc_feature    5871..7053
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of pAGT5888 - Fragment 2 (concatenated
                     sequence 6)"
     misc_feature    6325..6328
                     /note="Geneious type: ligation"
                     /label="Ligation"
     intron          6327..6462
                     /label="I9 (Intron Aratp11)"
     misc_feature    6329..6457
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_E2_(I9-UNI) - Fragment 2
                     (concatenated sequence 2)"
     misc_feature    6458..6461
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    6462..6821
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_E3_(E10) - Fragment 2
                     (concatenated sequence 3)"
     misc_feature    6822..6825
                     /note="Geneious type: ligation"
                     /label="Ligation"
     intron          6823..6943
                     /label="I10 (Intron e2f58)"
     misc_feature    6826..6936
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_E4_(I10-UNI) - Fragment 2
                     (concatenated sequence 4)"
     misc_feature    6937..6940
                     /note="Geneious type: ligation"
                     /label="Ligation"
     misc_feature    6941..>7053
                     /note="Geneious type: Concatenated sequence"
                     /label="Digestion of NbLbCpf1_E5_(E11) - Fragment 2
                     (concatenated sequence 5)"
     CDS             7004..7051
                     /label="Nucleoplasm NLS"
ORIGIN      
        1 ttcgtgagac cacgaagtgc cgaggcacga acccagtgga cataagcctg ttcggttcgt
       61 aagctgtaat gcaagtagcg tatgcgctca cgcaactggt ccagaacctt gaccgaacgc
      121 agcggtggta acggcgcagt ggcggttttc atggcttgtt atgactgttt ttttggggta
      181 cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg
      241 ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatc
      301 atgggggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc
      361 gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc
      421 ggcctgaagc cacacagcga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa
      481 acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc
      541 gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt
      601 tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt
      661 atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa
      721 catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag
      781 gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct
      841 ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc
      901 aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat
      961 cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc
     1021 tcgcgcgcag atcagttgga agaatttgtc cattacgtga aaggcgagat caccaaggta
     1081 gtcggcaaat aatgtctagc tagaaattcg ttcaagccga cgccgcttcg cggcgcggct
     1141 taactcaagc gttagatgca ctaagcacat aattgctcac agccaaacta tcaggtcaag
     1201 tctgctttta ttatttttaa gcgtgcataa taagccctac acaaattggg agatatatca
     1261 tgctgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     1321 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     1381 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     1441 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     1501 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     1561 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     1621 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     1681 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     1741 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     1801 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     1861 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     1921 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     1981 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     2041 ttttacggtt cctgcactct gtggtctcaa atgctgcagc ctaagaagaa gagaaaggtt
     2101 ggaggagtcg actcgagtgc ggccgccaca atgagcaagc tcgagaagtt taccaactgc
     2161 tacagcctgt ctaagaccct gaggttcaag gctattcctg tgggtaagac ccaagagaat
     2221 atcgacaaca agcggctgct ggttgaggat gagaagagag ctgaggatta caagggcgtg
     2281 aagaagctgc tggatcggta ctacctgagc ttcatcaacg atgtgctgca cagcatcaag
     2341 ctgaagaacc tgaacaacta catcagcctg ttccggaaga aaacccggac cgagaaagag
     2401 aacaaagagc ttgagaacct cgagatcaac ctgcggaaag agatcgctaa ggctttcaag
     2461 ggtaacgaag gtaaggattt ttatgatata ctatgcttat gtattttgta ctgaaagcat
     2521 atcctgcttc attgggatat tactgaaagc atttaactac atgtaaactc acttgatgat
     2581 caataaactt gattttgcag gttacaagag cctgttcaag aaggatatta tcgagactat
     2641 cctgcctgag ttcctggacg ataaggatga gattgccctg gtgaacagct tcaacggttt
     2701 cactactgcc ttcaccggtt tcttcagaaa ccgggaaaac atgttcagcg aagaggccaa
     2761 gtctacctct atcgctttcc ggtgcattaa cgagaacttg acccggtaca tcagcaacat
     2821 ggacatcttc gagaaggtgg acgccatctt cgataagcac gaggtgcaag aaatcaaaga
     2881 gaagatcctg aactccgact acgacgtcga ggattttttt gagggcgagt tcttcaactt
     2941 cgtgctcacc caagaaggta acattcctta gttacctttc ttttcttttt ccatcataag
     3001 tttatagatt gtacatgctt tgagattttt ctttgcaaac aatctcaggt atcgatgtgt
     3061 acaacgctat catcggtggt ttcgtgactg agagcggtga gaagattaag ggcctgaacg
     3121 agtacattaa cctgtacaat caaaagacca agcagaagct gccgaagttc aagccgcttt
     3181 acaagcaggt tctgagcgat cgtgagagcc tgtcttttta cggagaggga tacacctctg
     3241 atgaagaggt tttggaggta atcttgaaat tggaactctt cttttgttgt ctaaacctat
     3301 caatttcttt gcggaaattt atttgaagct gtagagttaa aattgagtct tttaaacttt
     3361 tgtaggtgtt ccgtaacacc ctgaacaaga acagcgagat cttcagctcc atcaagaagc
     3421 tggaaaagct gtttaagaac ttcgacgagt acagcagcgc tggcatcttc gttaagaacg
     3481 gtcctgctat cagcaccatc agcaaggata ttttcggcga gtggaacgtg atccgggata
     3541 agtggaatgc tgagtacgat gacatccacc tgaagaaaaa ggctgtggtg accgagaagt
     3601 acgaggatga taggcggaag tccttcaaga agataggtaa gttgttactt atgattgttt
     3661 tcctctctgc tacatgtatt ttgttgttca tttctgtaag atataagaat tgagttttcc
     3721 tctgatgata ttattaggtt cctttagcct cgagcagctt caagagtatg ctgacgctga
     3781 tctgtccgtg gtcgagaagc ttaaagagat catcatccag aaggtcgacg agatctacaa
     3841 ggtgtacggc agctctgaga agcttttcga tgctgacttc gtgttggaga agtctctgaa
     3901 gaagaacgac gccgttgtcg ctatcatgaa ggatctgctg gacagcgtga agtctttcga
     3961 gaactatatc aaggccttct tcggcgaagg ttagtatcat atgaagaaat acctagtttc
     4021 agttgatgaa tgctattttc tgacctcagt tgttctcttt tgagaattat ttcttttcta
     4081 atttgcctga tttttctatt aattcattag gtaaagagac taatagggac gagtcattct
     4141 acggcgattt cgtgctggct tacgacatcc ttcttaaggt ggaccacatc tacgacgcca
     4201 tcagaaatta cgtgacccag aagccgtaca gcaaggacaa gttcaagttg tacttccaga
     4261 atccgcagtt catgggcggc tgggacaaag acaaagagac agattacagg gctaccatcc
     4321 tgcggtacgg ctctaagtac taccttgcca tcatggacaa gaaatacgcc aagtgcctgc
     4381 aaaagatcga caaggatgat gtgaacggca actacgagaa gatcaactac aagctcctgc
     4441 caggtaaatc ctggtccaca cttttacgat aaaaacacaa gattttaaac tatgaactga
     4501 tcaataatca ttcctaaaag accacacttt tgttttgttt ctaaagtaat ttttactgtt
     4561 ataacaggtc ctaacaagat gcttcctaag gtgttcttct caaagaaatg gatggcctac
     4621 tacaacccga gcgaggacat ccagaaaatc tacaagaacg gcaccttcaa aaagggcgac
     4681 atgttcaacc tgaacgactg ccacaagctg atcgatttct tcaaggacag catcagccgg
     4741 tatccgaagt ggtctaacgc ttacgatttc aacttcagcg agactgagaa gtataaggat
     4801 atcgccggct tctaccgtga ggttgaggaa cagggttaca aggttagctt cgagagcgcc
     4861 agcaagaaag aggtggacaa gttggttgaa gaaggtaaga ggtcaaaagg tttccgcaat
     4921 gatccctctt tttttgtttc tctagtttca agaatttggg tatatgacta acttctgagt
     4981 gttccttgat gcatatttgt gatgagacaa atgtttgttc tatgttttag gtaagctgta
     5041 catgttccaa atctataaca aggacttctc cgacaagtct cacggcactc ctaatctgca
     5101 tacaatgtac ttcaagctgc tgttcgacga gaacaaccac ggtcagatta ggctttctgg
     5161 tggtgctgag ctgttcatga gaagggcctc actgaagaaa gaagagttgg tcgttcaccc
     5221 tgccaactct ccaatcgcta acaagaaccc tgacaacccg aaaaagacca ccaccttgtc
     5281 ttacgacgtg tacaaggata agcggttcag cgaggatcag tacgagcttc acattccgat
     5341 cgccatcaac aagtgcccga agaacatctt caagatcaat accgaggtgc gggtgctgct
     5401 gaagcacgat gataatcctt acgtgatcgg catcgatagg ggcgagagaa accttcttta
     5461 catcgtggtg gtggacggca agggcaatat cgttgagcag tactctctga acgagattat
     5521 caacaatttc aacggcatcc ggatcaagac cgactaccac tctctgctgg ataagaaaga
     5581 aaaagagcgg ttcgaggcca ggcagaactg gacttctatc gaaaacatca aagagctgaa
     5641 ggccggctac atctctcagg tggtgcataa gatttgcgag ctggtggaaa agtacgacgc
     5701 tgtgattgct ctccaggatc tgaacagcgg cttcaagaac tcacgtgtga aggtaaagca
     5761 actgtgtttt aatcaatttc ttgtcaggat atatggatta taacttaatt tttgagaaat
     5821 ctgtagtatt tggcgtgaaa tgagtttgct ttttggtttc tcccgtgtta taggttgaga
     5881 agcaggtcta ccaaaagttc gagaagatgc tcatcgacaa gctgaactac atggtggaca
     5941 aaaagagcaa cccttgcgct accggtggtg ctcttaaggg ttaccagatc actaacaagt
     6001 tcgagtcttt caagagcatg agcacccaga acggcttcat cttctacatc cctgcttggc
     6061 tgaccagcaa gatcgatcct tctactggct tcgtcaacct gctcaagacc aagtacacca
     6121 gcattgccga cagcaagaag ttcatcagct cattcgaccg gatcatgtac gtgccagaag
     6181 aggatctttt cgagttcgcc ctcgattaca agaacttctc taggaccgac gccgactaca
     6241 ttaagaagtg gaagctgtac tcctacggca accggattcg gatctttcgg aacccgaaga
     6301 aaaacaacgt gttcgactgg gaagaggtaa agtttccaac tttcctttac catatcaaac
     6361 tatagttcga aactttttat ttgatcaact tcaaggccac ccgatctttc tattcctgat
     6421 taatttgtga tgaatccata ttgacttttg atggttacgc aggtgtgcct gacctctgcc
     6481 tacaaagaac tgttcaacaa gtacggcatc aactaccagc agggtgatat tagggctctg
     6541 ctttgcgagc agtctgacaa ggctttctac agctctttca tggccctgat gtctctgatg
     6601 ctgcaaatga ggaactctat caccggtagg accgatgtgg acttccttat ctctccggtg
     6661 aagaacagtg acgggatctt ctacgacagc cggaattatg aggctcaaga gaacgcaatc
     6721 ctgccgaaga atgctgatgc taacggcgct tacaacattg ccagaaaggt gctgtgggct
     6781 atcggccagt ttaagaaagc cgaagatgag aagttggaca aggtctgtct ttcctatttc
     6841 atatgtttaa tcctaggaat ttgatcaatt gattgtatgt atgtcgatcc caagactttc
     6901 ttgttcactt atatcttaac tctctctttg ctgtttcttg caggtgaaga tcgctatctc
     6961 caacaaagag tggctcgagt acgctcagac tagcgttaag cataaaaggc cggcggccac
     7021 gaaaaaggcc ggccaggcaa aaaagaaaaa ggg
//
