LOCUS       sequence-169717-        7869 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty gRNA delivery vector for Bacillus subtilis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7869)
  AUTHORS   Westbrook AW, Moo-Young M, Chou CP
  TITLE     Development of a CRISPR-Cas9 toolkit for comprehensive engineering 
            of Bacillus subtilis.
  JOURNAL   Appl Environ Microbiol. 2016 Jun 3. pii: AEM.01159-16.
  PUBMED    27260361
REFERENCE   2  (bases 1 to 7869)
  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: "Appl 
            Environ Microbiol. 2016 Jun 3. pii: AEM.01159-16."
COMMENT     Sequence Label: pAW005-2
FEATURES             Location/Qualifiers
     source          1..7869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    62..83
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(200..217)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(371..959)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(451..470)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2216..2234)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     2409..2428
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2642..2664
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2656..2673
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2657..2673
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2683..2702
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2683..2701
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cgactgggga gagccgaaag ataaggaagt ctggtaagag ctctaaagcc tggggtgcct
       61 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
      121 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
      181 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
      241 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
      301 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
      361 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
      421 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
      481 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
      541 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
      601 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
      661 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
      721 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
      781 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
      841 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
      901 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
      961 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     1021 ggattttggt catgagatta tcaaaaagga tcttcaccta gatccttagc gcttctaggg
     1081 acctctttag ctccttggaa gctgtcagta gtatacctaa taatttatct acattccctt
     1141 tagtaacgtg taactttcca aatttacaaa agcgactcat agaattattt cctcccgtta
     1201 aataatagat aactattaaa aatagacaat acttgctcat aagtaacggt acttaaattg
     1261 tttactttgg cgtgtttcat tgcttgatga aactgatttt tagtaaacag ttgacgatat
     1321 tctcgattga cccattttga aacaaagtac gtatatagct tccaatattt atctggaaca
     1381 tctgtggtat ggcgggtaag ttttattaag acactgttta cttttggttt aggatgaaag
     1441 cattccgctg gcagcttaag caattgctga atcgagactt gagtgtgcaa gagcaaccct
     1501 agtgttcggt gaatatccaa ggtacgcttg tagaatcctt cttcaacaat cagatagatg
     1561 tcagacgcat ggctttcaaa aaccactttt ttaataattt gtgtgcttaa atggtaagga
     1621 atactcccaa caattttata cctctgtttg ttagggaatt gaaactgtag aatatcttgg
     1681 tgaattaaag tgacacgagt attcagtttt aatttttctg acgataagtt gaatagatga
     1741 ctgtctaatt caatagacgt tacctgttta cttattttag ccagtttcgt cgttaaatgc
     1801 cctttacctg ttccaatttc gtaaacggta tcggtttctt ttaaattcaa ttgttttatt
     1861 atttggttga gtactttttc actcgttaaa aagttttgag aatattttat atttttgttc
     1921 atgtaatcac tccttcttaa ttacaaattt ttagcatcta atttaacttc aattcctatt
     1981 atacaaaatt ttaagatact gcactatcaa cacactctta agtttgcttc taagtcttat
     2041 ttccataact tcttttacgt ttccgccatt ctttgctgtt tcgattttta tgatatggtg
     2101 caagtcagca cgaacacgaa ccgtcttatc tcccattata tctttttttg cactgattgg
     2161 tgtatcattt cgtttttctt tttgtgctag aggatcaatt cttgaagacg aaagggcctc
     2221 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     2281 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2341 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcagccggc gaacgtggcg
     2401 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     2461 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     2521 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     2581 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     2641 tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag
     2701 ggcgaattgg gtaccgggcc ccccctcgag ttaacctgca ggaagtcatg taaaagatga
     2761 ggttggttca ttctcgaaaa ttacatctgt gttctcagag cggggcgtga gctttgaaaa
     2821 aatccttcag ctgccaatta aaggccatga tgagttagct gaaatcgtaa ttgtcacaca
     2881 tcatacatca gaagctgatt tcagtgatat cctgcaaaac ctaaatgatt tggaagtcgt
     2941 tcaagaagtc aaaagcacat atcgtgtaga agggaacggt tggagctaat gtggaaagga
     3001 cttatccatc aatataaaga atttttacct gtaacagatc aaacaccggc gctaacttta
     3061 catgaaggaa acacacctct tattcacctg ccgaagctgt ctgagcagct cggaattgag
     3121 cttcatgtca aaacggaagg cgtcaatcct acgggatcat ttaaagatcg cggaatggtt
     3181 atggctgtgg caaaggcaaa agaagaaggc aatgacacga ttatgtgcgc gtcaacaggt
     3241 aacacttccg ctgctgcggc agcatatgca gcccgtgcta acatgaaatg cattgtcatc
     3301 atcccgaacg gaaaaattgc atttggaaaa ctcgctcaag ctgtcatgta cggagccgag
     3361 attatcgcaa ttgacggaaa ctttgacgat gcgcttaaaa ttgtccgttc catctgtgag
     3421 aaatcaccga ttgcccttgt caactcagtc aacccttacc gcattgaagg ccaaaaaact
     3481 gctgccttcg ctagccataa aaaactaaaa aaaatattga aaatactgac gaggttatat
     3541 aagatgcatg ctgtaactga tgtcaggatc cctacccaat cagtacgtta attttggctt
     3601 taatgagttg taattcctct ggggcaactg ttcctttctt cgttgctcct cttgcccgcc
     3661 aggcgatact ttttacctga tcagctaacg ctacgccatc acgttcctga ccggataaaa
     3721 caacttcgaa cggatatcct tttgattgcg ttgtacaagg aacacacaga cacatacctg
     3781 ttttgttgtt gtacatgaaa ggactcagga caacagctgg acgatgtcca gcttgctcgc
     3841 taccttttgt cgggtcaaaa tcaacccaaa tcagatcgcc catatcgggt acgtatcggc
     3901 ttaccatacc tgcttcctcc ttaagatcta tgttgagtaa agcgttttca tttaaacctt
     3961 caattgtacg tacttatatt aaaatttaac gaaaattaca tttaattagt acgtacaaat
     4021 atagaataat cttgtttgca ttttctgtca atgttttctt acaaagaacg ctgtgatata
     4081 ctgaaatttg tccgtataca ttttggagga atggatatgt taccaaaata cgcgcaagta
     4141 aaagaagaaa tcagttcttg gattaatcaa ggcaaaatac tgcccgatca aaaaatccct
     4201 accgaaaacg aattaatgca gcaattcggc gtcagccggc ataccatccg caaagcgatc
     4261 ggcgacctcg tatcacaagg tctgctgtac agcgtgcaag gcggaggcac ctttgtcgct
     4321 tcacgctctg ctaagtcagc gctgcattcc aataaaacga tcggtgtttt gacaacttac
     4381 atatcagact atattttccc gagcatcatc agaggaatcg agtcctattt aagcgagcag
     4441 gggtattcta tgcttttgac aagcacaaac aacaacccgg acaatgaaag aagaggctta
     4501 gaaaacctgc tgtcccagca tattgacgga ctcatcgtag aaccgacaaa aagcgccctt
     4561 caaaccccaa acatcggcta ttatctgaac ttggagaaaa acggcattcc ttttgcgatg
     4621 attaacgcgt catatgccga gcttgccgcg ccaagtttta ccttggatga tgtgaaaggc
     4681 gggatgatgg cggcggagca tttgctttct ctcggccaca cgcatatgat gggtattttt
     4741 aaagctgatg acacacaagg cgtgaaacgg atgaacggat ttatacaggc gcaccgggag
     4801 cgtgagttgt ttccttctcc ggatatgatc gtgacattta caacggaaga aaaagaatca
     4861 aaacttctgg agaaagtaaa agccacactg gagaaaaaca gcaagcacat gccgacagcc
     4921 attctttgtt ataacgatga aattgcgctg aaggtgattg atatgctgag ggagatggat
     4981 cttaaagtgc cggaggatat gtctattgtc gggtacgatg attcacattt cgcccaaatc
     5041 tcagaagtga aactaacctc tgtcaaacat ccgaaatcag tgcttggaaa agcagccgcc
     5101 aaatatgtca ttgactgctt agagcataaa aagccgaagc aagaggatgt catatttgag
     5161 cctgagttga tcattcgcca gtccgcacga aaactgaatg aataagaatt cctgcagctg
     5221 gcgaatggcg attttcgttc gtgaatacat gttataataa ctataactaa taacgtaacg
     5281 tgactggcaa gagatatttt taaaacaatg aataggttta cacttacttt agttttatgg
     5341 aaatgaaaga tcatatcata tataatctag aataaaatta actaaaataa ttattatcta
     5401 gataaaaaat ttagaagcca atgaaatcta taaataaact aaattaagtt tatttaatta
     5461 acaactatgg atataaaata ggtactaatc aaaatagtga ggaggatata tttgaataca
     5521 tacgaacaaa ttaataaagt gaaaaaaata cttcggaaac atttaaaaaa taaccttatt
     5581 ggtacttaca tgtttggatc aggagttgag agtggactaa aaccaaatag tgatcttgac
     5641 tttttagtcg tcgtatctga accattgaca gatcaaagta aagaaatact tatacaaaaa
     5701 attagaccta tttcaaaaaa aataggagat aaaagcaact tacgatatat tgaattaaca
     5761 attattattc agcaagaaat ggtaccgtgg aatcatcctc ccaaacaaga atttatttat
     5821 ggagaatggt tacaagagct ttatgaacaa ggatacattc ctcagaagga attaaattca
     5881 gatttaacca taatgcttta ccaagcaaaa cgaaaaaata aaagaatata cggaaattat
     5941 gacttagagg aattactacc tgatattcca ttttctgatg tgagaagagc cattatggat
     6001 tcgtcagagg aattaataga taattatcag gatgatgaaa ccaactctat attaacttta
     6061 tgccgtatga ttttaactat ggacacgggt aaaatcatac caaaagatat tgcgggaaat
     6121 gcagtggctg aatcttctcc attagaacat agggagagaa ttttgttagc agttcgtagt
     6181 tatcttggag agaatattga atggactaat gaaaatgtaa atttaactat aaactattta
     6241 aataacagat taaaaaaatt ataaaaaaat tgaaaaaatg gtggaaacac ttttttcaat
     6301 ttttttgttt tattatttaa tatttgggaa atattcattc taattggtaa tcagatttta
     6361 gaaaacaata aacccttgca tagggggatc tcgacatgga tgagcgatga tgatatccgt
     6421 ttaggctggg cggtgatagc ttctcgttca ggcagtacgc ctcttttctt ttccagacct
     6481 gagggaggcg gaaatggtgt gaggttggcg cgccctcgag gccttccgaa aatgcgcggc
     6541 tttgaagctg agggtgcggc ggcaatcgtg cgcaatgaag tgattgaaaa tccggaaaca
     6601 atagcgacag ccattcgtat cggaaacccg gcaagctggg acaaagctgt aaaggcagcc
     6661 gaggaatcca atgggaaaat tgacgaagtc actgatgatg aaatccttca cgcatatcag
     6721 ctgatcgccc gtgtagaagg cgtgtttgca gaaccaggtt cttgcgcgtc tatcgcagga
     6781 gtgctgaaac aggtgaaatc cggagaaatt ccgaaaggca gcaaggtcgt agctgtgtta
     6841 acaggaaacg gactgaaaga tccgaacaca gcggtcgaca tttcagaaat caagcctgtc
     6901 acattgccga ctgatgaaga cagcatcctt gaatatgtaa aaggagcggc ccgtgtatga
     6961 acgaagccga catgctgttc tctgtcactg ttcccggaag cacagctaac ctaggccccg
     7021 gctttgattc agtcggaatg gcgctcagca gatatttgaa gctgaccgtc tttgaaagcg
     7081 acaaatggtc ttttgaggct gaaacagaaa cagtcgccgg aattaccggt caattgttgc
     7141 tacattgact ttaccgcttg catttaagaa tttagggtca atttgttttt tgattttttc
     7201 agcagccgct gcatcatttt tagcagacat caaatacaag tctttaaggt tcgcatcatc
     7261 taaaataatg tttttcgggt agccattgcc gccctctttt ccaactgtca acaatgtcac
     7321 ttcaaactgg ctgaggaaat cttctggaga tgtattgctg ccgccgtttg ctttaaaatc
     7381 tccataatta agcgccatta gaaactagtc gataaccaga agcaataaaa aatcaaatcg
     7441 gatttcacta tataatctca ctttatctaa gatgaatccg atggaagcat cctgttttct
     7501 ctcaattttt ttatctaaaa cccagcgttc gatgcttctt tgagcgaacg atcaaaaata
     7561 agtgccttcc catcaaaaaa atattctcaa cataaaaaac tttgtgtaat acttgtaacg
     7621 ctacatggag attaactcaa tctagagggt attaataatg atccacagta gcgtaaagcg
     7681 ttggggaaat tcaccggcgg tgcggattcc ggctacgtta atgcaggcgc tcaatctgaa
     7741 tattgatgat gaagtgaaga ttgacctggt ggatggcaaa ttaattattg agccagtgcg
     7801 taaagagccc gtatttacgc ttgctgaact ggtcaacgac atcacgccgg aaaacctcca
     7861 cgagaatat
//
