LOCUS       sequence-434229-        6683 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  episomal (HygR), with constitutive mCherry (Psmyc) and AHT-inducible
            sgRNA cloning site, including Golden Gate for multiple sgRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6683)
  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 6683)
  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: "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-Cherry-sgRNA
FEATURES             Location/Qualifiers
     source          1..6683
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1069..1085)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1070..1086)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     rep_origin      5654..6242
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6143..6162
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6396..6413
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6530..6551
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6566..6571,6572..6589,6590..6596)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6604..6620
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     6609..6631
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6628..6644
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6628..6644
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tctagaagaa atattggatc gtcggcaccg tcacggccgt gggaggcggc acgatccgcg
       61 acgtgatgat cggccgcatc cccacggtgc tgcgcagtga gctctacgcc atcccggcgt
      121 tgatctgtgc gttcgcacgc acaggcccgg tgtgagaagg gtctctgacg agcgggagaa
      181 cccacccggg gtgggcgagt ttgtcctgcg tgtgctcggt cgagtaggct ctgggagtac
      241 ccgtgtgtac gaccagcacg gcatacatca tttcgacgcc gagagattcg ccgcccgaaa
      301 tgagcacgat ccgcatggta ccatcaggag gaatcacttc gcaggatcca tggtctcgaa
      361 gggcgaggag gacaacatgg cgatcatcaa ggagttcatg cgcttcaagg tccacatgga
      421 gggctcggtc aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccgtacga
      481 gggcacccag accgccaagc tgaaggtcac caagggcggc ccgctgccgt tcgcctggga
      541 catcctgtcg ccgcagttca tgtacggcag caaggcctac gtcaagcacc cggccgacat
      601 cccggactac ctgaagctgt cgttcccgga gggcttcaag tgggagcgcg tcatgaactt
      661 cgaggacggc ggcgtcgtca ccgtcaccca ggactcgtcg ctgcaggacg gcgagttcat
      721 ctacaaggtc aagctgcggg gcaccaactt cccgtcggac ggcccggtca tgcagaagaa
      781 gaccatgggc tgggaggcct cgtcggagcg catgtacccg gaggacggcg ccctgaaggg
      841 cgagatcaag cagcggctga agctgaagga cggcggccac tacgacgccg aggtcaagac
      901 cacctacaag gccaagaagc cggtccagct gccgggcgcc tacaacgtga acatcaagct
      961 ggacatcacc agccacaacg aggactacac catcgtcgag cagtacgagc gcgccgaggg
     1021 ccgccacagc accggcggca tggacgagct gtacaagtga aagcttatcg ataccgtcga
     1081 cctcgagggg gggcccccgg ggatcatcga gccgagaacg ttatcgaagt tggtcatgtg
     1141 taatcccctc gtttgaactt tggattaagc gtagatacac ccttggacaa gccagttgga
     1201 ttcggagaca agcaaattca gccttaaaaa gggcgaggcc ctgcggtggt ggaacaccgc
     1261 agggcctcta accgctcgac gcgctgcacc aaccagcccg cgaacggctg gcagccagcg
     1321 taaggcgcgg ctcatcgggc ggcgttcgcc acgatgtcct gcacttcgag ccaagcctcg
     1381 aacacctgct ggtgtgcacg actcacccgg ttgttgacac cgcgcgcggc cgtgcgggct
     1441 cggtggggcg gctctgtcgc ccttgccagc gtgagtagcg cgtacctcac ctcgcccaac
     1501 aggtcgcaca cagccgattc gtacgccata aagccaggtg agcccaccag ctccgtaagt
     1561 tcgggcgctg tgtggctcgt acccgcgcat tcaggcggca gggggtctaa cgggtctaag
     1621 gcggcgtgta cggccgccac agcggctctc agcggcccgg aaacgtcctc gaaacgacgc
     1681 atgtgttcct cctggttggt acaggtggtt gggggtgctc ggctgtcgct ggtgttccac
     1741 caccagggct cgacgggaga gcgggggagt gtgcagttgt ggggtggccc ctcagcgaaa
     1801 tatctgactt ggagctcgtg tcggaccata caccggtgat taatcgtggt ctactaccaa
     1861 gcgtgagcca cgtcgccgac gaatttgagc agctctggct gccgtactgg ccgctggcaa
     1921 gcgacgatct gctcgagggg atctaccgcc aaagccgcgc gtcggcccta ggccgccggt
     1981 acatcgaggc gaacccaaca gcgctggcaa acctgctggt cgtggacgta gaccatccag
     2041 acgcagcgct ccgagcgctc agcgcccggg ggtcccatcc gctgcccaac gcgatcgtgg
     2101 gcaatcgcgc caacggccac gcacacgcag tgtgggcact caacgcccct gttccacgca
     2161 ccgaatacgc gcggcgtaag ccgctcgcat acatggcggc gtgcgccgaa ggccttcggc
     2221 gcgccgtcga cggcgaccgc agttactcag gcctcatgac caaaaacccc ggccacatcg
     2281 cctgggaaac ggaatggctc cactcagatc tctacacact cagccacatc gaggccgagc
     2341 tcggcgcgaa catgccaccg ccgcgctggc gtcagcagac cacgtacaaa gcggctccga
     2401 cgccgctagg gcggaattgc gcactgttcg attccgtcag gttgtgggcc tatcgtcccg
     2461 ccctcatgcg gatctacctg ccgacccgga acgtggacgg actcggccgc gcgatctatg
     2521 ccgagtgcca cgcgcgaaac gccgaattcc cgtgcaacga cgtgtgtccc ggaccgctac
     2581 cggacagcga ggtccgcgcc atcgccaaca gcatttggcg ttggatcaca accaagtcgc
     2641 gcatttgggc ggacgggatc gtggtctacg aggccacact cagtgcgcgc cagtcggcca
     2701 tctcgcggaa gggcgcagca gcgcgcacgg cggcgagcac agttgcgcgg cgcgcaaagt
     2761 ccgcgtcagc catggaggca ttgctatgag cgacggctac agcgacggct acaaccggca
     2821 gccgactgtc cgcaaaaagc ggcgcgtgac cgccgccgaa ggcgctcgaa tcaccggact
     2881 atccgaacgc cacgtcgtcc ggctcgtggc gcaggaacgc agcgagtggc tcgccgagca
     2941 ggctgcacgc cgcgaacgca tccgcgccta tcacgacgac gagggccact cttggccgca
     3001 aacggccaaa catttcgggc tgcatctgga caccgttaag cgactcggct atcgggcgag
     3061 gaaagagcgt gcggcagaac aggaagcggc tcaaaaggcc cacaacgaag ccgacaatcc
     3121 accgctgttc taacgcaatt ggggagcggg tgtcgcgggg gttccgtggg gggttccgtt
     3181 gcaacgggtc ggacaggtaa aagtcctggt agacgctagt tttctggttt gggccatgcc
     3241 tgtctcgttg cgtgtttcgt tgcgcccgtt ttgaatacca gccagacgtg gctccctcac
     3301 gatatcaata aacgaaaggc tcagtcgaaa gactgggcct ttcgtttatc tgttgtttga
     3361 aaaaaaaaag cgccgcaact gcggcgcttt tttttttgaa ttctctgacc agggaaaata
     3421 gccctctgac ctggggattt gcgatcccta tcagtgatag atataatctg ggaggagacg
     3481 attaatgcgt ctcggttttt gtactcgaaa gaagctacaa agataaggct tcatgccgaa
     3541 atcaacaccc tgtcatttta tggcagggtg tttttttttt gtcgacttgg ggaccctaga
     3601 ggtccccttt tttatttttt tggatgaaga gcattatcaa tggctcttca caggaagctt
     3661 aaaaaaaaaa gccccgcgat tgcggggctt ttttttttta atacggttat ccacagaatc
     3721 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3781 aaaggctggc cactctcgtt gcacggttcg ttgtgccgtt acaggccccg ttgacagctc
     3841 accgaacgta gttaaaacat gctggtcaaa ctaggtttac caacgatacg agtcagctca
     3901 tctagggcca gttctaggcg ttgttcgttg cgcggttcgt tgcgcatgtt tcgtgtggtt
     3961 gctagatggc tccgcaacca cacgcttcga ggttgagtgc ttccagcacg ggcgcgatcc
     4021 agaagaactt cgtcgtgcga ctgtcctcgt tgatccttgc cgagctggga tggaagcccg
     4081 gccgacccac cctggaggag atgatcgagg atgccagggc ctttcacgcc cgccgctgct
     4141 gagcgtccgc cgccgggccc gcaccgccgt cggccggccc gctccgggct cgcagcagcg
     4201 ggcttcggcg cgggcccggg gctcccgggc cgccgggcgg ggctccgccc ggcggccgcc
     4261 gggggccggg ggcggcgccg ggcggcccgg ggcgtcaggc gccgggggcg gtgtccggcg
     4321 gcccccagag gaactgcgcc agttcctccg gatcggtgaa gccggagaga tccagcgggg
     4381 tctcctcgaa cacctcgaag tcgtgcagga aggtgaaggc gagcagttcg cgggcgaagt
     4441 cctcggtccg cttccactgc gccccgtcga gcagcgcggc caggatctcg cggtcgcccc
     4501 ggaaggcgtt gagatgcagt tgcaccaggc tgtagcggga gtctcccgca tagacgtcgg
     4561 tgaagtcgac gatcccggtg acctcggtcg cggccaggtc cacgaagatg ttggtcccgt
     4621 gcaggtcgcc gtggacgaac cggggttcgc ggccggccag cagcgtgtcc acgtccggca
     4681 gccagtcctc caggcggtcc agcagccggg gcgagaggta gccccacccg cggtggtcct
     4741 cgacggtcgc cgcgcggcgt tcccgcagca gttccgggaa gacctcggaa tggggggtga
     4801 gcacggtgtt cccggtcagc ggcaccctgt gcagccggcc gagcacccgg ccgagttcgc
     4861 gggccagggc gagcagcgcg ttccggtcgg tcgtgccgtc catcgcggac cgccaggtgg
     4921 tgccggtcat ccggctcatc accaggtagg gccacggcca ggctccggtg ccgggccgca
     4981 gctcgccgcg gccgaggagg cggggcaccg gcaccggggc gtccgccagg accgcgtacg
     5041 cctccgactc cgacgcgagg ctctccggac cgcaccagtg ctcgccgaac agcttgatca
     5101 ccgggccggg ctcgccgacc agtacggggt tggtgctctc gccgggcacc cgcagcaccg
     5161 gcggcaccgg cagcccgagc tcctccaggg ctcggcgggc cagcggctcc cagaattcct
     5221 ggtcgttccg caggctcgcg taggaatcat ccgaatcaat acggtcgaga agtaacaggg
     5281 attcttgtgt cacagcggac ctctattcac agggtacggg ccggcttaat tccgcacggc
     5341 cggtcgcgac acggcctgtc cgcaccgcgg atcaggcgtt gacgatgacg ggctggtcgg
     5401 ccacgtcggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5461 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5521 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5581 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5641 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5701 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5761 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5821 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5881 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5941 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6001 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6061 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6121 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6181 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6241 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6301 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6361 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     6421 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6481 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     6541 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     6601 gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt accagatctg
     6661 gctcgcaccg cggtggcggc cgc
//
