LOCUS       sequence-440182-        8881 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Cas9 under control of AHT inducible TetR on integrating plasmid 
            (KanR) using L5 integrase and attP for site-specific integration 
            into attB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8881)
  AUTHORS   Neo DM, Clatworthy AE, Hung DT
  TITLE     A dual-plasmid CRISPR/Cas9-based method for rapid and efficient 
            genetic disruption in Mycobacterium abscessus.
  JOURNAL   J Bacteriol. 2024 Mar 21;206(3):e0033523. doi: 10.1128/jb.00335-23. 
            Epub 2024 Feb 6.
  PUBMED    38319218
REFERENCE   2  (bases 1 to 8881)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J 
            Bacteriol."; date: "2024-03-21"; volume: "206(3):e0033523. doi"; 
            pages: " 10.1128/jb.00335-23. Epub 2024 Feb 6"
COMMENT     Sequence Label: pDN-Sth1Cas9
FEATURES             Location/Qualifiers
     source          1..8881
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(496..1084)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(576..595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1267..1285
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1297..1315
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      4795..4822
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             5212..5835
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(5285..5304)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(7838..7857)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             7849..8664
                     /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(7922..7941)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 aataaacgaa aggctcagtc gaaagactgg gcctttcgtt tatctgttgt ttgaaaaaaa
       61 aaagcgccgc aactgcggcg cttttttttt tgaattctct gaccagggaa aatagccctc
      121 tgacctgggg atttgcgatc cctatcagtg atagatataa tctgggagga gacgattaat
      181 gcgtctcggt ttttgtactc gaaagaagct acaaagataa ggcttcatgc cgaaatcaac
      241 accctgtcat tttatggcag ggtgtttttt ttttgtcgac ttggggaccc tagaggtccc
      301 cttttttatt tttttggatg aagagcatta tcaatggctc ttcacaggaa gcttaaaaaa
      361 aaaagccccg cgattgcggg gctttttttt tttaatacgg ttatccacag aatcagggga
      421 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      481 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      541 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      601 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      661 tctcccctcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      721 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      781 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      841 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      901 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
      961 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1021 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     1081 tcaagaagat cctttgatct tttctacggg gtctgacgct catggaacaa taaacgaaag
     1141 gctcagtcga aagactgggc ctttcgttta tctgttgttt gaaaaaaaaa agccccgcga
     1201 ttgcggggct tttttttttc atatgtctga ccagggaaaa tagccctctg acctggggat
     1261 ttgacttccc tatcagtgat agagataatc tgggagtccc tatcagtgat agagaaggcg
     1321 gtatcgatat gtcggacctg gtgctgggcc tggacatcgg catcggctcg gtgggcgtgg
     1381 gcatcctgaa caaggtgacc ggcgagatca tccacaagaa ctcgcgcatc ttcccggccg
     1441 cccaggccga gaacaacctg gtgcgccgca ccaaccgcca gggccgccgc ctggcccgcc
     1501 gcaagaagca ccgccgcgtg cgcctgaacc gcctgttcga ggagtcgggc ctgatcaccg
     1561 acttcaccaa gatctcgatc aacctgaacc cgtaccagct gcgcgtgaag ggcctgaccg
     1621 acgagctgtc gaacgaggag ctgttcatcg ccctgaagaa catggtgaag caccgcggca
     1681 tctcgtacct ggacgacgcc tcggacgacg gcaactcgtc ggtgggcgac tacgcccaga
     1741 tcgtgaagga gaactcgaag cagctggaga ccaagacccc gggccagatc cagctggagc
     1801 gctaccagac ctacggccag ctgcgcggcg acttcaccgt ggagaaggac ggcaagaagc
     1861 accgcctgat caacgtgttc ccgacctcgg cctaccgctc ggaggccctg cgcatcctgc
     1921 agacccagca ggagttcaac ccgcagatca ccgacgagtt catcaaccgc tacctggaga
     1981 tcctgaccgg caagcgcaag tactaccacg gcccgggcaa cgagaagtcg cgcaccgact
     2041 acggccgcta ccgcacctcg ggcgagaccc tggacaacat cttcggcatc ctgatcggca
     2101 agtgcacctt ctacccggac gagttccgcg ccgccaaggc ctcgtacacc gcccaggagt
     2161 tcaacctgct gaacgacctg aacaacctga ccgtgccgac cgagaccaag aagctgtcga
     2221 aggagcagaa gaaccagatc atcaactacg tgaagaacga gaaggccatg ggcccggcca
     2281 agctgttcaa gtacatcgcc aagctgctgt cgtgcgacgt ggccgacatc aagggctacc
     2341 gcatcgacaa gtcgggcaag gccgagatcc acaccttcga ggcctaccgc aagatgaaga
     2401 ccctggagac cctggacatc gagcagatgg accgcgagac cctggacaag ctggcctacg
     2461 tgctgaccct gaacaccgag cgcgagggca tccaggaggc cctggagcac gagttcgccg
     2521 acggctcgtt ctcgcagaag caggtggacg agctggtgca gttccgcaag gccaactcgt
     2581 cgatcttcgg caagggctgg cacaacttct cggtgaagct gatgatggag ctgatcccgg
     2641 agctgtacga gacctcggag gagcagatga ccatcctgac ccgcctgggc aagcagaaga
     2701 ccacctcgtc gtcgaacaag accaagtaca tcgacgagaa gctgctgacc gaggagatct
     2761 acaacccggt ggtggccaag tcggtgcgcc aggccatcaa gatcgtgaac gccgccatca
     2821 aggagtacgg cgacttcgac aacatcgtga tcgagatggc ccgcgagacc aacgaggacg
     2881 acgagaagaa ggccatccag aagatccaga aggccaacaa ggacgagaag gacgccgcca
     2941 tgctgaaggc cgccaaccag tacaacggca aggccgagct gccgcactcg gtgttccacg
     3001 gccacaagca gctggccacc aagatccgcc tgtggcacca gcagggcgag cgctgcctgt
     3061 acaccggcaa gaccatctcg atccacgacc tgatcaacaa ctcgaaccag ttcgaggtgg
     3121 accacatcct gccgctgtcg atcaccttcg acgactcgct ggccaacaag gtgctggtgt
     3181 acgccaccgc caaccaggag aagggccagc gcaccccgta ccaggccctg gactcgatgg
     3241 acgacgcctg gtcgttccgc gagctgaagg ccttcgtgcg cgagtcgaag accctgtcga
     3301 acaagaagaa ggagtacctg ctgaccgagg aggacatctc gaagttcgac gtgcgcaaga
     3361 agttcatcga gcgcaacctg gtggacaccc gctacgcctc gcgcgtggtg ctgaacgccc
     3421 tgcaggagca cttccgcgcc cacaagatcg acaccaaggt gtcggtggtg cgcggccagt
     3481 tcacctcgca gctgcgccgc cactggggca tcgagaagac ccgcgacacc taccaccacc
     3541 acgccgtgga cgccctgatc atcgccgcct cgtcgcagct gaacctgtgg aagaagcaga
     3601 agaacaccct ggtgtcgtac tcggaggacc agctgctgga catcgagacc ggcgagctga
     3661 tctcggacga cgagtacaag gagtcggtgt tcaaggcccc gtaccagcac ttcgtggaca
     3721 ccctgaagtc gaaggagttc gaggactcga tcctgttctc gtaccaggta gactcgaagt
     3781 tcaaccgcaa gatctcggac gccaccatct acgccacccg ccaggccaag gtgggcaagg
     3841 acaaggccga cgagacctac gtgctgggca agatcaagga catctacacc caggacggct
     3901 acgacgcctt catgaagatc tacaagaagg acaagtcgaa gttcctgatg taccgccacg
     3961 acccgcagac cttcgagaag gtgatcgagc cgatcctgga gaactacccg aacaagcaga
     4021 tcaacgacaa gggcaaggag gtgccgtgca acccgttcct gaagtacaag gaggagcacg
     4081 gctacatccg caagtactcg aagaagggca acggcccgga gatcaagtcg ctgaagtact
     4141 acgactcgaa gctgggcaac cacatcgaca tcaccccgaa ggactcgaac aacaaggtgg
     4201 tgctgcagtc ggtgtcgccg tggcgcgccg acgtgtactt caacaagacc accggcaagt
     4261 acgagatcct gggcctgaag tacgccgacc tgcagttcga caagggcacc ggcacctaca
     4321 agatctcgca ggagaagtac aacgacatca agaagaagga gggcgtggac tcggactcgg
     4381 agttcaagtt caccctgtac aagaacgacc tgctgctggt gaaggacacc gagaccaagg
     4441 agcagcagct gttccgcttc ctgtcgcgca ccatgccgaa gcagaagcac tacgtggagc
     4501 tgaagccgta cgacaagcag aagttcgagg gcggcgaggc cctgatcaag gtgctgggca
     4561 acgtggccaa ctcgggccag tgcaagaagg gcctgggcaa gtcgaacatc tcgatctaca
     4621 aggtgcgcac cgacgtgctg ggcaaccagc acatcatcaa gaacgagggc gacaagccga
     4681 agctggactt ctaaatactc tggccatttt tctttaccgc ctttgagtga gctgataccg
     4741 ctcgccgcag ccgaacgacc gagcgcaacg cgtgcggccg ctgattagct aagcagaagg
     4801 ccatcctgac ggatggcctt tttctagaaa aaaaaaaagc gccgcaactg cggcgctttt
     4861 ttttttttaa ttaaagaaat attggatcgt cggcaccgtc acggccgtgg gaggcggcac
     4921 gatccgcgac gtgatgatcg gccgcatccc cacggtgctg cgcagtgagc tctacgccat
     4981 cccggcgttg atctgtgcgt tcgcacgcac aggcccggtg tgagaagggt ctctgacgag
     5041 cgggagaacc cacccggggt gggcgagttt gtcctgcgtg tgctcggtcg agtaggctct
     5101 gggagtaccc gtgtgtacga ccagcacggc atacatcatt tcgacgccga gagattcgcc
     5161 gcccgaaatg agcacgatcc gcatggtacc atcaggagga atcacttcgc aatgtctaga
     5221 ttagataaaa gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa
     5281 ggtttaacaa cccgtaaact cgcccagaag cttggtgtag agcagcctac attgtattgg
     5341 catgtaaaaa ataagcgggc cctgctcgac gccttagcca ttgagatgtt agataggcac
     5401 catactcact tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct
     5461 aaaagtttta gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca
     5521 cggcctacag aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa
     5581 ggtttttcac tagagaacgc gttatatgca ctcagcgctg tggggcattt taccttaggt
     5641 tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact
     5701 gatagtatgc cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag
     5761 ccagccttct tattcggcct tgaattgatc atctgcggat tagaaaaaca acttaaatgt
     5821 gaaagtgggt cttaatgacc atgggctagc gctttagcta attaatttaa aataaaaaag
     5881 gggacctcta gggtccccaa ttaaatagtt aacactagta ttaaaaaaaa aagccccgcg
     5941 attgcggggc tttttttttt cgacggcgag ggttccgacc gctgcaactc ccggtgcaac
     6001 cttgtcccgg tctattctct tcactgcacc agctccaatc tggtgtgaat gcccctcgtc
     6061 tgttcgcgca ggcggggggc tctattcgtt tgtcagcatc gaaagtagcc agatcaggga
     6121 tgcgttgcaa ccgcgtatgc ccaggtcaga agagtcgcac aagagttgca gacccctgga
     6181 aagaaaaatg gccagagggc gaaaacaccc tctgaccagc ggagcgggcg acgggaatcg
     6241 aacccgcgta gctagtttgg aagaatgggt gtctgccgac cacatatatg ggccggtcaa
     6301 gataggtttt taccccctct cggctgcatc ctctaagtgg aaagaaattg caggtcgtag
     6361 aagcgcgttg aagcctgaga gttgcacagg agttgcaacc cggtagcctt gttcacgacg
     6421 agaggagacc tagttggcac gtcgcggatg gggatcgctg aagactcagc gcagcgggag
     6481 gatccaagcc tcatacgtca acccgcagga cggtgtgagg tactacgcgc tgcagaccta
     6541 cgacaacaag atggacgccg aagcctggct cgcgggcgag aagcggctca tcgagatgga
     6601 gacctggacc cctccacagg accgggcgaa gaaggcagcc gccagcgcca tcacgctgga
     6661 ggagtacacc cggaagtggc tcgtggagcg cgacctcgca gacggcacca gggatctgta
     6721 cagcgggcac gcggagcgcc gcatctaccc ggtgctaggt gaagtggcgg tcacagagat
     6781 gacgccagct ctggtgcgtg cgtggtgggc cgggatgggt aggaagcacc cgactgcccg
     6841 ccggcatgcc tacaacgtcc tccgggcggt gatgaacaca gcggtcgagg acaagctgat
     6901 cgcagagaac ccgtgccgga tcgagcagaa ggcagccgat gagcgcgacg tagaggcgct
     6961 gacgcctgag gagctggaca tcgtcgccgc tgagatcttc gtgcactacc ggatcgcggc
     7021 atacatcctg gcgtggacga gcctccggtt cggagagctg atcgagcttc gccgcaagga
     7081 catcgtggac gacggcatga cgatgaagct ccgggtgcgc cgtggcgctt cccgcgtggg
     7141 gaacaagatc gtcgttggca acgccaagac cgtccggtcg aagcgtcctg tgacggttcc
     7201 gcctcacgtc gcggagatga tccgagcgca catgaaggac cgtacgaaga tgaacaaggg
     7261 ccccgaggca ttcctggtga ccacgacgca gggcaaccgg ctgtcgaagt ccgcgttcac
     7321 caagtcgctg aagcgtggct acgccaagat cggtcggccg gaactccgca tccacgacct
     7381 ccgcgctgtc ggcgctacgt tcgccgctca ggcaggtgcg acgaccaagg agctgatggc
     7441 ccgcctcggt cacacgactc ctaggatggc gatgaagtac cagatggcgt ctgaggcccg
     7501 cgacgaggct atcgctgagg cgatgtccaa gctggccaag acctcctgaa acgcaaaaag
     7561 cccccctccc aaggacactg agtcctaaag aggggggttt cttgtcagta cgcgaagaac
     7621 cacgcctggc cgcgagcgcc agcaccgccg ctctgtgcgg agacctgggc accagccccg
     7681 ccgccgccag gagcattgcc gttcccgcca gctagccacc tgacgtcggg ggggggggaa
     7741 agccacgttg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     7801 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     7861 caacgggaaa cgtcttgctc gaggccgcga ttaaattcca acatggatgc tgatttatat
     7921 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
     7981 gggaagcccc atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     8041 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     8101 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa
     8161 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     8221 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     8281 cgcgtatttc gcctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     8341 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataat
     8401 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     8461 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     8521 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     8581 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     8641 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc
     8701 attacgctga cttgacggga cggcggcttt gttgaataaa tcgaactttt gctgagttga
     8761 aggatcagat cacgcatctt cccgacaacg cagaccgttc cgtggcaaag caaaagttca
     8821 aaatcaccaa ctggtccacc tacaacaaag ctctcaccaa ccgtggctcc ctcacgatat
     8881 c
//
