LOCUS       sequence-403323-        9288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one editing CRISPR-Cas construct for one-step genome editing 
            of Streptomyces using AsCas12j-2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9288)
  AUTHORS   Tan LL, Heng E, Zulkarnain N, Leong CY, Ng V, Yang LK, San Seow DC, 
            Peh G, Lim YH, Lokanand K, Kanagasundaram YH, Ng SB, Wong FT
  TITLE     Application of Cas12j for Streptomyces editing and cluster 
            activation
  JOURNAL   bioRxiv 2021.10.28.465406
REFERENCE   2  (bases 1 to 9288)
  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: "bioRxiv 
            2021.10.28.465406"
COMMENT     Sequence Label: pCRISPomyces-AsCas12j-2
FEATURES             Location/Qualifiers
     source          1..9288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..1598)
                     /codon_start=1
                     /gene="rep"
                     /product="replication initiator protein from the 
                     Streptomyces ghanaensis plasmid pSG5"
                     /label=pSG5 Rep
                     /translation="MWKPGEATWGNTCRCNNVHTCPWCMSRILAVRGSNVQLAADGLAD
                     AGYGLHLGTNTLRHFERMAFGTVRKGMRHGLVAVLHDGWKGAYGSSGRRWRTMRDDFGI
                     IGYERAFEDTFGWGSGWHLHWHTLWVTREVLGPDAQAAFRDALAGAWAAGVESAGGYTV
                     SETCDRPGCSCEGKGHGTDVRPLNGADAADGDAGKQARYLYKDGDKTKGGVAKIGLELA
                     GQNFKAGRGDDRMGPLDLGDAAAAELQRLRRPGPFVEKYREREFGVFQVRKHYRSQNLN
                     RLIKELGIQQDVRTEEEITDDTEGLVAIAVIPAYIWYRYIARVAGRRLDLIKVAETYGL
                     PGVRRLVESWGLVWGKDVLDPPAPEAPAAPGDLDADQMRFEVMSEEEAAFREARRKANE
                     ARTEELAASLDRVRQPKKEAIRPTISLRKRLKPKPVTVDVKTPPPGAASPVCRRCKGKL
                     APVLQPWGQHPGDCLRVDTAVA"
     oriT            2765..2874
                     /note="incP origin of transfer"
     promoter        3096..3397
                     /label=rpsL promoter
                     /note="promoter for the Xylanimonas cellulosilytica 
                     ribosomal protein S12 gene"
     promoter        5871..6155
                     /label=gapdh promoter
                     /note="glyceraldehyde-3-phosphate dehydrogenase promoter 
                     from Eggerthella lenta"
     CDS             complement(6200..6496)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSG"
     primer_bind     6346..6368
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6360..6377
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6361..6377
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6387..6406
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6387..6405
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6453..6473)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6453..6471)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6492..6508)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6492..6508)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6505..6527)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6516..6532
                     /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)."
     promoter        complement(join(6540..6546,6547..6564,6565..6570))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6585..6606
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     terminator      6665..6699
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     rep_origin      complement(6913..7501)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6993..7012)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             7705..8505
                     /codon_start=1
                     /gene="aac(3)IV"
                     /product="aminoglycoside 3-N-acetyltransferase type IV     
                               "
                     /label=ApmR
                     /note="confers resistance to apramycin (Paget and Davies, 
                     1996)"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
ORIGIN
        1 cggcggacgc tcggactggg cgcgggattc cagcagtaac ccaggtccgc cggccacctc
       61 acggcaggca gaccctcggc tttcgcgccg ggaccgccat gagaccgcca cccggatgtc
      121 cggggtggcg gtctcatggc ggtccctcag cggccctacg cgaccgctgt gtcgacgcgg
      181 aggcagtctc cggggtgctg gccccagggc tggagtaccg gggcgagctt gcccttgcag
      241 cggcggcaca cgggcgacgc ggcgccgggc gggggagtct tgacgtcgac ggtgaccggc
      301 ttcggcttca ggcgcttccg aaggctgatc gtcgggcgga tcgcctcctt cttcggctga
      361 cgcactcggt cgagcgaagc ggccagctcc tccgtgcggg cctcgttggc cttccggcgc
      421 gcctcgcgga aagcagcttc ctcctcggac atgacctcga acctcatctg gtcagcgtca
      481 aggtcgcccg gcgcggccgg cgcttccggg gcgggcgggt ccaggacgtc cttgccccac
      541 accaggcccc aggactcgac gagccgccgg acgcccggta ggccgtacgt ctcggcgacc
      601 ttgatgaggt cgaggcgacg tccggcgacg cgggcgatgt atcggtacca gatgtaggcc
      661 gggatgaccg cgatggcgac caggccctcg gtgtcgtcgg tgatctcctc ctcggtgcgg
      721 acgtcctgct ggatgccgag ttccttgatc agccggttca ggttctgcga ccggtagtgc
      781 ttgcggacct ggaacacgcc gaactcgcgc tcgcggtact tctcgacgaa cgggccgggc
      841 cgacgaagcc gctgcagctc ggcggccgcc gcgtcgccca ggtcgagcgg tcccatgcgg
      901 tcgtcgccgc gaccggcctt gaagttctgt ccggccagct ccaggccgat cttggcgacg
      961 ccgcccttgg tcttgtcgcc gtccttgtag aggtagcggg cctgcttgcc cgcatcgccg
     1021 tcagcggcgt ccgcgccgtt gagtgggcgc acgtcggtgc cgtggccctt gccctcacag
     1081 gagcaaccgg gccggtcgca cgtctcgctg acggtgtagc cgcccgcgga ttcgaccccg
     1141 gcggcccagg ctccggcgag tgcgtcgcgg aacgcggcct gggcgtccgg gccgagcacc
     1201 tcgcgggtga cccagagcgt gtgccagtgc aggtgccagc cggagcccca gccgaaggtg
     1261 tcctcgaagg cccgctcgta gccgatgatc ccgaagtcgt cgcgcatcgt gcgccagcgg
     1321 cggccggacg agccgtacgc gcccttccag ccgtcgtgca agaccgcgac caggccgtgc
     1381 cgcattccct tgcggacggt gccgaacgcc atgcgctcga agtggcgcaa cgtgttcgtg
     1441 ccaaggtgca gcccgtaccc ggcgtccgcg agaccgtcgg cggcgagctg cacgttcgag
     1501 ccccgtacgg ccaggatgcg gctcatgcac cacgggcagg tgtggacgtt gttgcagcgg
     1561 cacgtgttgc cccacgtcgc ctcgcccggc ttccacatca gctcggccgt cccggcagtg
     1621 agccgggtcc cgcagccctt gaacgcctcg ttcagcgaca ccgtctggtg ccggtcccgc
     1681 cgggcgaacc gctcgtcgcg cgggtcctcc cgcccggctg tcgcggcacc ctcgtttggg
     1741 gtagaacccg ttccagttac agcgctctga cctgcagtgg acggagattt tcccttacta
     1801 ctaaagcccg cgtccggatt acccgctgta gtcgtgcttg ctacgctgcg tgactggtcc
     1861 gcaatgagac gctttgcgcg ctttcggcag gcgtccgagc agtagatttt ggggcgcttc
     1921 ccggggatgt ggacgatcgg ggtgccgcag tggcacttcg gtccggcggg gcgcggtggt
     1981 gtcgacgcgc tgttctctcg tacgctcgtc acagagcaaa cgtcctcact cggcatgctg
     2041 cgccggttcg ggggcggcga gcccgggagg ccaatcccgg gctcgtgcca tttctgggtc
     2101 ctgttgatcc tggcattggt gtggccgttc attgcccctg ctcgctcctg acgcgccgat
     2161 agacgtccga tacgcccggt gctggtggga tttgataggt cggaagaagc cccgcccggg
     2221 cctgggcggg gcttcctgtg cgtcaggacc tcctcgtcgt gagcctcttc ggcctatgga
     2281 cggagtgacc tcgtgatccg ttacagccgc gcgcgctcgc gtagagcggt ctcatcagtt
     2341 ccacgaacgg tcctcttcgc agatcagggc gttggggcgg agtctcacca aggactacgt
     2401 ctgctggcga tttccgttac accccgggcg gtggccggcg cacacgcgcg cccgcgttgg
     2461 gcagtgcaga aagtgcagaa acctaggcgc tgatggtcca ggtccacggt tcgtcgtcgg
     2521 cggcggcgcg ggcggcggcg tcggccaggg cgcgggcgag accggctacg gcgggcttga
     2581 tgcgccggtt gcgggcgacc ttgagcagct agtatgcagg tcgacggatc ttttccgctg
     2641 cataaccctg cttcggggtc attatagcga ttttttcggt atatccatcc tttttcgcac
     2701 gatatacagg attttgccaa agggttcgtg tagactttcc ttggtgtatc caacggcgtc
     2761 agccgggcag gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc ttcactgtcc
     2821 cttattcgca cctggcggtg ctcaacggga atcctgctct gcgaggctgg ccggctaccg
     2881 ccggcgtaac agatgagggc aagcggatgg ctgatgaaac caagccaacc aggaagggca
     2941 gcccacctat caaggtgtac tgccttccag acgaacgaag agcgattgag gaaaaggcgg
     3001 cggcggccgg catgagcctg tcggcctacc tgctggccgt cggccagggc tacaaaatca
     3061 cgggcgtcgt ggactatgag cacgtccgcg agctggccct gcaggcggaa gtcaggtaga
     3121 cacgacttcc gctagtcctt gcaaggtctg ctgacgtgag gcggggcggt cgtttttgac
     3181 cgccctgcct tcgtcatgta ggctcgctcg ctgtgcctgg cgtgtcatca gacgcccagg
     3241 tcccggtgcc gtgaggcccg ggccatcgag ccggtggtac gtggctgcgg tccccttgtg
     3301 agggctgcgc gccgtgtgct gtccggcgcg cacagccttg aatccacccg cgggggccgg
     3361 ccggtctccg tgagctcgag tagacgacgg agacgtaatg cccaagcccg cggtggagag
     3421 cgagttcagc aaggtcctca agaagcactt ccccggcgag cggttccggt cgtcctacat
     3481 gaagcgtggc ggcaagatcc tggcggccca gggcgaggaa gccgtggtgg cgtacctgca
     3541 gggcaagtcg gaggaagagc cgccgaactt ccagccgccg gccaagtgcc acgtggtgac
     3601 caagtcccgc gacttcgcgg agtggccgat catgaaggcc tccgaggcga tccagcgcta
     3661 catctacgcc ctgtccacca ccgagcgcgc ggcctgcaag ccgggcaagt cgtccgagtc
     3721 ccacgcggcc tggttcgcgg ccaccggcgt ctccaaccac ggctactcgc acgtgcaggg
     3781 cctgaacctg atcttcgacc acaccctggg ccgctacgac ggcgtcctga agaaggtgca
     3841 gctgcgcaac gagaaggccc gcgcccgcct ggagtccatc aacgcctcgc gcgccgacga
     3901 gggcctgccg gagatcaagg ccgaggaaga ggaagtcgcg accaacgaga cgggccacct
     3961 gctgcagccg ccgggcatca acccgtcctt ctacgtgtac cagaccatct cgccgcaggc
     4021 ctaccgcccg cgcgacgaga tcgtgctgcc gccggagtac gcgggctacg tgcgcgaccc
     4081 gaacgccccg atcccgctgg gcgtcgtgcg caaccgctgc gacatccaga agggctgccc
     4141 gggctacatc ccggagtggc agcgcgaggc gggcaccgcc atctccccca agaccggcaa
     4201 ggccgtcacc gtgccgggcc tgtcgccgaa gaagaacaag cgcatgcgcc gctactggcg
     4261 ctccgagaag gagaaggccc aggacgccct gctggtcacc gtgcgcatcg gcaccgactg
     4321 ggtggtgatc gacgtgcgcg gcctgctgcg caacgcccgc tggcgcacca tcgcgccgaa
     4381 ggacatctcg ctgaacgccc tgctggacct gttcaccggc gacccggtca tcgacgtgcg
     4441 tcgcaacatc gtgaccttca cctacaccct ggacgcctgc ggcacctacg cccgcaagtg
     4501 gaccctgaag ggcaagcaga ccaaggccac cctggacaag ctgaccgcca cccagaccgt
     4561 ggccctggtg gcgatcgacc tgggccagac caacccgatc tcggcgggca tctcgcgcgt
     4621 cacccaggag aacggcgccc tgcagtgcga gccgctggac cgcttcaccc tgccggacga
     4681 cctgctgaag gacatctccg cctaccgcat cgcctgggac cgcaacgagg aagagctgcg
     4741 cgcccgctcc gtcgaggcgc tgccggaggc ccagcaggcc gaggtccgcg ccctggacgg
     4801 cgtgtcgaag gagacggccc gcacccagct gtgcgccgac ttcggcctgg acccgaagcg
     4861 cctgccgtgg gacaagatgt cgtccaacac caccttcatc tccgaggccc tgctgtcgaa
     4921 ctccgtctcg cgcgaccagg tgttcttcac cccggccccg aagaagggcg ccaagaagaa
     4981 ggccccggtc gaggtgatgc gcaaggaccg cacctgggcc cgcgcctaca agccgcgcct
     5041 gtccgtcgag gcccagaagc tgaagaacga ggccctgtgg gccctgaagc gcacctcgcc
     5101 ggagtacctg aagctgtcgc gccgcaagga agagctgtgc cgccgctcga tcaactacgt
     5161 catcgaaaag acccgtcgcc gcacccagtg ccagatcgtc atcccggtga tcgaggacct
     5221 gaacgtgcgc ttcttccacg gctcgggcaa gcgcctgccg ggctgggaca acttcttcac
     5281 cgccaagaag gagaaccgct ggttcatcca gggcctgcac aaggcgttct cggacctgcg
     5341 cacccaccgc tcgttctacg tgttcgaggt gcgcccggag cgcacctcga tcacctgccc
     5401 gaagtgcggc cactgcgagg tgggcaaccg cgacggcgag gccttccagt gcctgtcctg
     5461 cggcaagacc tgcaacgcgg acctggacgt ggccacccac aacctgaccc aggtggcgct
     5521 gaccggcaag accatgccga agcgcgagga gccgcgcgac gcccagggca ccgccccggc
     5581 gcgcaagacc aagaaggcgt ccaagtcgaa ggccccgccc gccgagcgcg aggaccagac
     5641 cccggcccag gagcccagcc agaccagctg agaattcaga tctacgcgtt ccccgcaaaa
     5701 gcggcctttg actccctgca agcctcagcg accgaatata tcggttatgc gtgggcgatg
     5761 gttgttgtca ttgtcggcgc aactatcggt atcaagctgt ttaagaaatt cacctcgaaa
     5821 gcaagctgat aaaccgatac aattaaaggc tccttttgga gccttttttt gctgctcctt
     5881 cggtcggacg tgcgtctacg ggcaccttac cgcagccgtc ggctgtgcga cacggacgga
     5941 tcgggcgaac tggccgatgc tgggagaagc gcgctgctgt acggcgcgca ccgggtgcgg
     6001 agcccctcgg cgagcggtgt gaaacttctg tgaatggcct gttcggttgc tttttttata
     6061 cggctgccag ataaggcttg cagcatctgg gcggctaccg ctatgatcgg ggcgttcctg
     6121 caattcttag tgcgagtatc tgaaagggga tacgcgtcgg aacgctcaac gattgcccct
     6181 cacgagggga caagtcttct cagccgctac agggcgcgtc ccattcgcca ttcaggctgc
     6241 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     6301 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     6361 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg
     6421 ggccccccct cgaggtcctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
     6481 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     6541 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     6601 acattaattg cgttgcgctc actgcccgct ttccaccggt gaagacatac tccatctgga
     6661 tttgttcaga acgctcggtt gccgccgggc gttttttatc tagaaggtaa taagaactac
     6721 acgactggat actgactttt cacactagtc ataactcccc cagtcctgca cgctgtcgta
     6781 ttctcctggc cacgacttta caacaccgca cagcatgttg tcaaagcaga gaccgttcga
     6841 atgtgaacaa agcttcatat gcatgctagc tctagaggcc aggaaccgta aaaaggccgc
     6901 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6961 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     7021 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     7081 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     7141 ggtcgttcgc tccaagctgg gctgtgtgca cgaccccccc gttcagcccg accgctgcgc
     7201 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     7261 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     7321 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     7381 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     7441 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     7501 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     7561 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt tggttcatgt
     7621 gcagctccat cagcaaaagg ggatgataag tttatcacca ccgactattt gcaacagtgc
     7681 cgttgatcgt gctatgatcg actgatgtca tcagcggtgg agtgcaatgt cgtgcaatac
     7741 gaatggcgaa aagccgagct catcggtcag cttctcaacc ttggggttac ccccggcggt
     7801 gtgctgctgg tccacagctc cttccgtagc gtccggcccc tcgaagatgg gccacttgga
     7861 ctgatcgagg ccctgcgtgc tgcgctgggt ccgggaggga cgctcgtcat gccctcgtgg
     7921 tcaggtctgg acgacgagcc gttcgatcct gccacgtcgc ccgttacacc ggaccttgga
     7981 gttgtctctg acacattctg gcgcctgcca aatgtaaagc gcagcgccca tccatttgcc
     8041 tttgcggcag cggggccaca ggcagagcag atcatctctg atccattgcc cctgccacct
     8101 cactcgcctg caagcccggt cgcccgtgtc catgaactcg atgggcaggt acttctcctc
     8161 ggcgtgggac acgatgccaa cacgacgctg catcttgccg agttgatggc aaaggttccc
     8221 tatggggtgc cgagacactg caccattctt caggatggca agttggtacg cgtcgattat
     8281 ctcgagaatg accactgctg tgagcgcttt gccttggcgg acaggtggct caaggagaag
     8341 agccttcaga aggaaggtcc agtcggtcat gcctttgctc ggttgatccg ctcccgcgac
     8401 attgtggcga cagccctggg tcaactgggc cgagatccgt tgatcttcct gcatccgcca
     8461 gaggcgggat gcgaagaatg cgatgccgct cgccagtcga ttggctgagc tcatgagcgg
     8521 agaacgagat gacgttggag gggcaaggtc gcgctgattg ctggggcaac acgtggagcg
     8581 gatcggggat tgtctttctt cagctcgctg atgatatgct gacgctcaat gccgtttggc
     8641 ctccgactaa cgaaaatccc gcatttggac ggctgatccg attggcacgg cggacggcga
     8701 atggcggagc agacgctcgt ccgggggcaa tgagatatga aaaagcctga actcaccgcg
     8761 acgtatcgat gtcgaggttc ctcaggggag ccaccccaga gaagccctcg gagctgagcg
     8821 gagctatttc caaagccatg ccagctagag acagtgcaca ccgccaagca tctgcaaaac
     8881 cctcgcctgg agggaaagtg caatgtacgt acgctggttt ccctccagaa agggattctg
     8941 cggcttctca actttggaag aagaggcgga acgaactgct gtttcagcct actcatgtga
     9001 ggaggctggt cctttaccct gatgcccgag ggcatcaggg taaaggacca gcctcgccaa
     9061 ctaggagcgc taccaaggcg aacacaaccc aggacttggt agaacccttc gccggagaag
     9121 ttcagactca tcggcatgag cacccctcgc cgcgcactcg gcaccggtcc gtcgaccacc
     9181 accaggacgt cgttgtcgac gtcggccccg cggctcctgc ccgccgaacg cgtcgtcgtc
     9241 gacggcctag tgctcatcga cgagcacccg gagccaggtg aaaagcgc
//
