LOCUS       sequence-201663-        8617 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR system used to genome editing in Mycobacterium smegmatis, 
            expresses Fn Cpf1 and crRNA targeting ligD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8617)
  AUTHORS   Sun B, Yang J, Yang S, Ye RD, Chen D, Jiang Y
  TITLE     A CRISPR-Cpf1-Assisted Non-Homologous End Joining Genome Editing 
            System of Mycobacterium smegmatis.
  JOURNAL   Biotechnol J. 2018 Sep;13(9):e1700588. doi: 10.1002/biot.201700588. 
            Epub 2018 Aug 6.
  PUBMED    30039929
REFERENCE   2  (bases 1 to 8617)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Biotechnol 
            J."; date: "2018-09"; volume: "13(9):e1700588. doi"; pages: " 
            10.1002/biot.201700588. Epub 2018 Aug 6"
COMMENT     Sequence Label: pSBY3_ligD
FEATURES             Location/Qualifiers
     source          1..8617
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..53)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(145..230)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(438..457)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             449..1264
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(522..541)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1595..2183
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2084..2103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      8549..8595
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gctagaaaga gtttgtagaa acgcaaaaag gccatccgtc aggatggcct tctgcttaat
       61 ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc gttgcttcgc
      121 aacgttcaaa tccgctcccg gcggatttgt cctactcagg agagcgttca ccgacaaaca
      181 acagataaaa cgaaaggccc agtctttcga ctgagccttt cgttttattt aaatcaccgt
      241 tgcgtgtgac gtgctatcta caacagtaga aattcggatc cattatacct aggactgagc
      301 tagctgtcaa tctagccacc tgacgtcggg ggggggggaa agccacgttg tgtctcaaaa
      361 tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc
      421 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc
      481 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
      541 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccc atgcgccaga
      601 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
      661 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
      721 tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga
      781 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
      841 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
      901 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
      961 tggctggcct gttgaacaag tctggaaaga aatgcataat cttttgccat tctcaccgga
     1021 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     1081 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     1141 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     1201 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     1261 ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga
     1321 cggcggcttt gttgaataaa tcgaactttt gctgagttga aggatcagat cacgcatctt
     1381 cccgacaacg cagaccgttc cgtggcaaag caaaagttca aaatcaccaa ctggtccacc
     1441 tacaacaaag ctctcaccaa ccgtggctcc ctcactttct ggctggatga tggggcgatt
     1501 caggcctggt atgagtcagc aacaccttct tcacgaggca gacctcacta gttccatgag
     1561 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     1621 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     1681 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     1741 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     1801 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     1861 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     1921 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     1981 gtgagctatg agaaagcgcc acgcttcccg aggggagaaa ggcggacagg tatccggtaa
     2041 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2101 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2161 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     2221 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     2281 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcaacg
     2341 cgtgagccca ccagctccgt aagttcgggc gctgtgtggc tcgtacccgc gcattcaggc
     2401 ggcagggggt ctaacgggtc taaggcggcg tgtacggccg ccacagcggc tctcagcggc
     2461 ccggaaacgt cctcgaaacg acgcatgtgt tcctcctggt tggtacaggt ggttgggggt
     2521 gctcggctgt cgctggtgtt ccaccaccag ggctcgacgg gagagcgggg gagtgtgcag
     2581 ttgtggggtg gcccctcagc gaaatatctg acttggagct cgtgtcggac catacaccgg
     2641 tgattaatcg tggtctacta ccaagcgtga gccacgtcgc cgacgaattt gagcagctct
     2701 ggctgccgta ctggccgctg gcaagcgacg atctgctcga ggggatctac cgccaaagcc
     2761 gcgcgtcggc cctaggccgc cggtacatcg aggcgaaccc aacagcgctg gcaaacctgc
     2821 tggtcgtgga cgtagaccat ccagacgcag cgctccgagc gctcagcgcc cgggggtccc
     2881 atccgctgcc caacgcgatc gtgggcaatc gcgccaacgg ccacgcacac gcagtgtggg
     2941 cactcaacgc ccctgttcca cgcaccgaat acgcgcggcg taagccgctc gcatacatgg
     3001 cggcgtgcgc cgaaggcctt cggcgcgccg tcgatggcga ccgcagttac tcaggcctca
     3061 tgaccaaaaa ccccggccac atcgcctggg aaacggaatg gctccactca gatctctaca
     3121 cactcagcca catcgaggcc gagctcggcg cgaacatgcc accgccgcgc tggcgtcagc
     3181 agaccacgta caaagcggct ccgacgccgc tagggcggaa ttgcgcactg ttcgattccg
     3241 tcaggttgtg ggcctatcgt cccgccctca tgcggatcta cctgccgacc cggaacgtgg
     3301 acggactcgg ccgcgcgatc tatgccgagt gccacgcgcg aaacgccgaa tttccgtgca
     3361 acgacgtgtg tcccggaccg ctaccggaca gcgaggtccg cgccatcgcc aacagcattt
     3421 ggcgttggat cacaaccaag tcgcgcattt gggcggacgg gatcgtggtc tacgaggcca
     3481 cactcagtgc gcgccagtcg gccatctcgc ggaagggcgc agcagcgcgc acggcggcga
     3541 gcacagttgc gcggcgcgca aagtccgcgt cagccatgga ggcattgcta tgagcgacgg
     3601 ctacagcgac ggctacaacc ggcagccgac tgtccgcaaa aagcggcgcg tgaccgccgc
     3661 cgaaggcgct cgaatcaccg gactatccga acgccacgtc gtccggctcg tggcgcagga
     3721 acgcagcgag tggctcgccg agcaggctgc acgccgcgaa cgcatccgcg cctatcacga
     3781 cgacgagggc cactcttggc cgcaaacggc caaacatttc gggctgcatc tggacaccgt
     3841 taagcgactc ggctatcggg cgaggaaaga gcgtgcggca gaacaggaag cggctcaaaa
     3901 ggcccacaac gaagccgaca atccaccgct gttctaacgc aattggggag cgggtgtcgc
     3961 gggggttccg tggggggttc cgttgcaacg ggtcggacag gtaaaagtcc tggtagacgc
     4021 tagttttctg gtttgggcca tgcctgtctc gttgcgtgtt tcgttgcgcc cgttttgaat
     4081 accagccaga cgagacgggg ttctacgaat cttggtcgat accaagccat ttccgctgaa
     4141 tatcggggag ctcaccgcca gaatcggtgg ttgtggtgat gtacgtggcg aactccgttg
     4201 tagtgcctgt ggtggcatcc gtggcgcggc cgcggtacca gatctttaaa tctagacggt
     4261 gaccacaacg cgcccgcttt gatcggggac gtctgcggcc gaccatttac gggtcttgtt
     4321 gtcgttggcg gtcatgggcc gaacatactc acccggatcg gagggccgag gacaaggtcg
     4381 aacgaggggc atgacccggt gcggggcttc ttgcactcgg cataggcgag tgctaagaat
     4441 aacgttggca ctcgcgaccg gtgagtgcta ggtcgggacg gtgaggccag gcccgtcgtc
     4501 gcagcgagtg gcagcgagga caacttgagc cgtccgtcgc gggcactgcg cccggccagc
     4561 gtaagtagcg gggttgccgt cacccggtga cccccgtttc atccccgatc cggaggaatc
     4621 acttcgcaat gtccatctac caagagtttg tgaataaata ctccctgtcc aagaccctcc
     4681 gttttgagct gatcccccaa ggcaagaccc tcgaaaacat caaggcacgc ggcctcatcc
     4741 tggatgacga aaagcgcgct aaggattaca agaaggcaaa gcagatcatc gacaagtacc
     4801 accagttctt catcgaagag atcctgtcct ccgtgtgcat ctccgaggac ctgctccaga
     4861 actactccga tgtctacttc aagctcaaga agtccgatga cgataacctg cagaaggact
     4921 tcaagtccgc taaggatacc atcaagaagc agatctccga atacatcaag gattccgaga
     4981 agttcaagaa cctcttcaac cagaacctga tcgacgcaaa gaagggccag gaatccgatc
     5041 tcatcctgtg gctcaagcag tccaaggata acggcatcga gctcttcaag gccaactccg
     5101 acatcaccga catcgatgaa gctctggaga tcatcaagtc cttcaagggc tggaccacct
     5161 acttcaaggg cttccacgaa aaccgcaaga acgtgtactc ctccaacgat atcccaacct
     5221 ctatcatcta ccgcatcgtc gacgataacc tgccaaagtt cctcgaaaac aaggcaaagt
     5281 acgagtccct gaaggataag gccccagaag ctatcaacta cgagcagatc aagaaggacc
     5341 tggccgaaga gctcaccttc gacatcgatt acaagacctc tgaagtgaac cagcgcgtct
     5401 tctccctcga tgaagtgttc gagatcgcca acttcaacaa ctacctgaac cagtccggca
     5461 tcaccaagtt caacaccatc atcggcggca agttcgtcaa cggcgaaaac accaagcgca
     5521 agggcatcaa cgagtacatc aacctctact cccagcagat caacgataag accctgaaga
     5581 agtacaagat gtccgtgctc ttcaagcaga tcctgtccga caccgaatcc aagtccttcg
     5641 tcatcgacaa gctggaggac gattccgatg tggtcaccac catgcagtcc ttctacgaac
     5701 agatcgcagc cttcaagacc gtggaagaga agtccatcaa ggagaccctc tccctgctct
     5761 tcgacgatct gaaggctcag aagctggatc tctccaagat ctacttcaag aacgacaagt
     5821 ccctgaccga tctctcccag caggtcttcg acgattactc cgtgatcggc accgcagtcc
     5881 tggaatacat cacccagcag atcgccccaa agaacctcga taacccatcc aagaaggaac
     5941 aggagctgat cgccaagaag accgaaaagg ctaagtacct gtccctcgag accatcaagc
     6001 tggctctcga agagttcaac aagcaccgcg acatcgataa gcagtgccgc ttcgaagaga
     6061 tcctcgcaaa cttcgctgca atcccaatga tcttcgacga aatcgcacag aacaaggata
     6121 acctggccca gatctccatc aagtaccaga accagggcaa gaaggatctg ctccaggcct
     6181 ccgctgagga cgatgtgaag gcaatcaagg acctgctcga tcagaccaac aacctgctcc
     6241 acaagctgaa gatcttccac atctcccagt ccgaagacaa ggccaacatc ctcgacaagg
     6301 atgagcactt ctacctggtg ttcgaagagt gctacttcga actcgctaac atcgtcccac
     6361 tgtacaacaa gatccgcaac tacatcaccc agaagccata ctccgatgaa aagttcaagc
     6421 tcaacttcga gaactccacc ctggcaaacg gctgggacaa gaacaaggaa ccagataaca
     6481 ccgccatcct cttcatcaag gacgataagt actacctggg cgtgatgaac aagaagaaca
     6541 acaagatctt cgacgataag gccatcaagg aaaacaaggg cgagggctac aagaagatcg
     6601 tgtacaagct gctcccaggc gctaacaaga tgctcccaaa ggtcttcttc tccgcaaagt
     6661 ccatcaagtt ctacaaccca tccgaagata tcctgcgcat ccgcaaccac tccacccaca
     6721 ccaagaacgg ctccccacag aagggctacg aaaagttcga gttcaacatc gaagactgcc
     6781 gcaagttcat cgatttctac aagcagtcca tctccaagca cccagagtgg aaggacttcg
     6841 gcttccgctt ctccgatacc cagcgctaca actccatcga tgaattctac cgcgaagtgg
     6901 agaaccaggg ctacaagctg accttcgaaa acatctccga gtcctacatc gattccgtgg
     6961 tcaaccaggg caagctgtac ctcttccaga tctacaacaa ggacttctcc gcttactcca
     7021 agggccgccc aaacctgcac accctctact ggaaggcact cttcgacgaa cgcaacctgc
     7081 aggatgtggt ctacaagctc aacggcgaag cagagctgtt ctaccgcaag cagtccatcc
     7141 caaagaagat cacccaccca gccaaggaag caatcgccaa caagaacaag gataacccaa
     7201 agaaggaatc cgtgttcgag tacgacctga tcaaggataa gcgcttcacc gaggacaagt
     7261 tcttcttcca ctgcccaatc accatcaact tcaagtcctc cggcgccaac aagttcaacg
     7321 atgaaatcaa cctgctcctg aaggagaagg ctaacgacgt gcacatcctg tccatcgatc
     7381 gcggcgaacg ccacctcgcc tactacaccc tggtcgacgg caagggcaac atcatcaagc
     7441 aggacacctt caacatcatc ggcaacgatc gcatgaagac caactaccac gacaagctgg
     7501 ccgctatcga gaaggaccgc gattccgctc gcaaggattg gaagaagatc aacaacatca
     7561 aggaaatgaa ggaaggctac ctctcccagg tggtccacga aatcgctaag ctggtgatcg
     7621 agtacaacgc aatcgtggtc ttcgaagacc tgaacttcgg cttcaagcgc ggccgcttca
     7681 aggtggagaa gcaggtctac cagaagctgg aaaagatgct catcgagaag ctgaactacc
     7741 tcgtgttcaa ggacaacgaa ttcgataaga ccggcggcgt cctccgtgca taccagctga
     7801 ccgccccatt cgagaccttc aagaagatgg gcaagcagac cggcatcatc tactacgtgc
     7861 cagctggctt cacctctaag atctgcccag tgaccggctt cgtcaaccag ctctacccaa
     7921 agtacgaatc cgtctccaag tcccaggagt tcttctccaa gttcgacaag atctgctaca
     7981 acctggataa gggctacttc gaattctcct tcgactacaa gaacttcggc gataaggcag
     8041 ccaagggcaa gtggaccatc gcatccttcg gctcccgcct catcaacttc cgcaactccg
     8101 acaagaacca caactgggat acccgcgaag tgtacccaac caaggaactg gagaagctcc
     8161 tgaaggatta ctccatcgaa tacggccacg gcgagtgcat caaggctgca atctgcggcg
     8221 aatccgacaa gaagttcttc gcaaagctga cctctgtgct caacaccatc ctgcagatgc
     8281 gcaactccaa gaccggcacc gagctggatt acctcatctc cccagtggcc gacgtcaacg
     8341 gcaacttctt cgattcccgc caggctccaa agaacatgcc acaggacgct gatgcaaacg
     8401 gcgcctacca catcggtctg aagggtctca tgctcctggg tcgcatcaag aacaaccagg
     8461 aaggcaagaa gctgaatctc gtcattaaga acgaagaata ctttgaattt gtccagaacc
     8521 gcaataacta agatcgactg ccaggcatca aataaaacga aaggctcagt cgaaagactg
     8581 ggcctttcgt tttatgccat catggccgcg gtgatca
//
