LOCUS       sequence-169719-        7974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multi-gRNA delivery vector containing ugtP-gRNA.P395T for Bacillus 
            subtilis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7974)
  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 7974)
  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: pAW014-2
FEATURES             Location/Qualifiers
     source          1..7974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     155..174
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     388..410
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     402..419
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     403..419
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     429..448
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        429..447
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        1334..1409
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    5782..5803
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5920..5937)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6091..6679)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6171..6190)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(7936..7954)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
       61 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
      121 aaatgcttca gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
      181 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
      241 atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc
      301 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
      361 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
      421 agcgcgcgta atacgactca ctatagggcg aattgggtac cgggcccccc ctcgagttaa
      481 cctgcaggaa gtcatgtaaa agatgaggtt ggttcattct cgaaaattac atctgtgttc
      541 tcagagcggg gcgtgagctt tgaaaaaatc cttcagctgc caattaaagg ccatgatgag
      601 ttagctgaaa tcgtaattgt cacacatcat acatcagaag ctgatttcag tgatatcctg
      661 caaaacctaa atgatttgga agtcgttcaa gaagtcaaaa gcacatatcg tgtagaaggg
      721 aacggttgga gctaatgtgg aaaggactta tccatcaata taaagaattt ttacctgtaa
      781 cagatcaaac accggcgcta actttacatg aaggaaacac acctcttatt cacctgccga
      841 agctgtctga gcagctcgga attgagcttc atgtcaaaac ggaaggcgtc aatcctacgg
      901 gatcatttaa agatcgcgga atggttatgg ctgtggcaaa ggcaaaagaa gaaggcaatg
      961 acacgattat gtgcgcgtca acaggtaaca cttccgctgc tgcggcagca tatgcagccc
     1021 gtgctaacat gaaatgcatt gtcatcatcc cgaacggaaa aattgcattt ggaaaactcg
     1081 ctcaagctgt catgtacgga gccgagatta tcgcaattga cggaaacttt gacgatgcgc
     1141 ttaaaattgt ccgttccatc tgtgagaaat caccgattgc ccttgtcaac tcagtcaacc
     1201 cttaccgcat tgaaggccaa aaaactgctg ccttcgctag ctgaatcata cagatctcat
     1261 aaaaaactaa aaaaaatatt gaaaatactg acgaggttat ataagatgca tgcaaggaaa
     1321 aactgctgga gatgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     1381 aacttgaaaa agtggcaccg agtcggtgct ttttttccat ggctacccaa tcagtacgtt
     1441 aattttggct ttaatgagtt gtaattcctc tggggcaact gttcctttct tcgttgctcc
     1501 tcttgcccgc caggcgatac tttttacctg atcagctaac gctacgccat cacgttcctg
     1561 accggataaa acaacttcga acggatatcc ttttgattgc gttgtacaag gaacacacag
     1621 acacatacct gttttgttgt tgtacatgaa aggactcagg acaacagctg gacgatgtcc
     1681 agcttgctcg ctaccttttg tcgggtcaaa atcaacccaa atcagatcgc ccatatcggg
     1741 tacgtatcgg cttaccatac ctgcttcctc cttaggatct atgttgagta aagcgttttc
     1801 atttaaacct tcaattgtac gtacttatat taaaatttaa cgaaaattac atttaattag
     1861 tacgtacaaa tatagaataa tcttgtttgc attttctgtc aatgttttct tacaaagaac
     1921 gctgtgatat actgaaattt gtccgtatac attttggagg aatggatatg ttaccaaaat
     1981 acgcgcaagt aaaagaagaa atcagttctt ggattaatca aggcaaaata ctgcccgatc
     2041 aaaaaatccc taccgaaaac gaattaatgc agcaattcgg cgtcagccgg cataccatcc
     2101 gcaaagcgat cggcgacctc gtatcacaag gtctgctgta cagcgtgcaa ggcggaggca
     2161 cctttgtcgc ttcacgctct gctaagtcag cgctgcattc caataaaacg atcggtgttt
     2221 tgacaactta catatcagac tatattttcc cgagcatcat cagaggaatc gagtcctatt
     2281 taagcgagca ggggtattct atgcttttga caagcacaaa caacaacccg gacaatgaaa
     2341 gaagaggctt agaaaacctg ctgtcccagc atattgacgg actcatcgta gaaccgacaa
     2401 aaagcgccct tcaaacccca aacatcggct attatctgaa cttggagaaa aacggcattc
     2461 cttttgcgat gattaacgcg tcatatgccg agcttgccgc gccaagtttt accttggatg
     2521 atgtgaaagg cgggatgatg gcggcggagc atttgctttc tctcggccac acgcatatga
     2581 tgggtatttt taaagctgat gacacacaag gcgtgaaacg gatgaacgga tttatacagg
     2641 cgcaccggga gcgtgagttg tttccttctc cggatatgat cgtgacattt acaacggaag
     2701 aaaaagaatc aaaacttctg gagaaagtaa aagccacact ggagaaaaac agcaagcaca
     2761 tgccgacagc cattctttgt tataacgatg aaattgcgct gaaggtgatt gatatgctga
     2821 gggagatgga tcttaaagtg ccggaggata tgtctattgt cgggtacgat gattcacatt
     2881 tcgcccaaat ctcagaagtg aaactaacct ctgtcaaaca tccgaaatca gtgcttggaa
     2941 aagcagccgc caaatatgtc attgactgct tagagcataa aaagccgaag caagaggatg
     3001 tcatatttga gcctgagttg atcattcgcc agtccgcacg aaaactgaat gaataagaat
     3061 tcctgcagct ggcgaatggc gattttcgtt cgtgaataca tgttataata actataacta
     3121 ataacgtaac gtgactggca agagatattt ttaaaacaat gaataggttt acacttactt
     3181 tagttttatg gaaatgaaag atcatatcat atataatcta gaataaaatt aactaaaata
     3241 attattatct agataaaaaa tttagaagcc aatgaaatct ataaataaac taaattaagt
     3301 ttatttaatt aacaactatg gatataaaat aggtactaat caaaatagtg aggaggatat
     3361 atttgaatac atacgaacaa attaataaag tgaaaaaaat acttcggaaa catttaaaaa
     3421 ataaccttat tggtacttac atgtttggat caggagttga gagtggacta aaaccaaata
     3481 gtgatcttga ctttttagtc gtcgtatctg aaccattgac agatcaaagt aaagaaatac
     3541 ttatacaaaa aattagacct atttcaaaaa aaataggaga taaaagcaac ttacgatata
     3601 ttgaattaac aattattatt cagcaagaaa tggtaccgtg gaatcatcct cccaaacaag
     3661 aatttattta tggagaatgg ttacaagagc tttatgaaca aggatacatt cctcagaagg
     3721 aattaaattc agatttaacc ataatgcttt accaagcaaa acgaaaaaat aaaagaatat
     3781 acggaaatta tgacttagag gaattactac ctgatattcc attttctgat gtgagaagag
     3841 ccattatgga ttcgtcagag gaattaatag ataattatca ggatgatgaa accaactcta
     3901 tattaacttt atgccgtatg attttaacta tggacacggg taaaatcata ccaaaagata
     3961 ttgcgggaaa tgcagtggct gaatcttctc cattagaaca tagggagaga attttgttag
     4021 cagttcgtag ttatcttgga gagaatattg aatggactaa tgaaaatgta aatttaacta
     4081 taaactattt aaataacaga ttaaaaaaat tataaaaaaa ttgaaaaaat ggtggaaaca
     4141 cttttttcaa tttttttgtt ttattattta atatttggga aatattcatt ctaattggta
     4201 atcagatttt agaaaacaat aaacccttgc atagggggat ctcgacatgg atgagcgatg
     4261 atgatatccg tttaggctgg gcggtgatag cttctcgttc aggcagtacg cctcttttct
     4321 tttccagacc tgagggaggc ggaaatggtg tgaggttggc gcgccctcga ggccttccga
     4381 aaatgcgcgg ctttgaagct gagggtgcgg cggcaatcgt gcgcaatgaa gtgattgaaa
     4441 atccggaaac aatagcgaca gccattcgta tcggaaaccc ggcaagctgg gacaaagctg
     4501 taaaggcagc cgaggaatcc aatgggaaaa ttgacgaagt cactgatgat gaaatccttc
     4561 acgcatatca gctgatcgcc cgtgtagaag gcgtgtttgc agaaccaggt tcttgcgcgt
     4621 ctatcgcagg agtgctgaaa caggtgaaat ccggagaaat tccgaaaggc agcaaggtcg
     4681 tagctgtgtt aacaggaaac ggactgaaag atccgaacac agcggtcgac atttcagaaa
     4741 tcaagcctgt cacattgccg actgatgaag acagcatcct tgaatatgta aaaggagcgg
     4801 cccgtgtatg aacgaagccg acatgctgtt ctctgtcact gttcccggaa gcacagctaa
     4861 cctaggcccc ggctttgatt cagtcggaat ggcgctcagc agatatttga agctgaccgt
     4921 ctttgaaagc gacaaatggt cttttgaggc tgaaacagaa acagtcgccg gaattaccgg
     4981 tcaattgttg ctacattgac tttaccgctt gcatttaaga atttagggtc aatttgtttt
     5041 ttgatttttt cagcagccgc tgcatcattt ttagcagaca tcaaatacaa gtctttaagg
     5101 ttcgcatcat ctaaaataat gtttttcggg tagccattgc cgccctcttt tccaactgtc
     5161 aacaatgtca cttcaaactg gctgaggaaa tcttctggag atgtattgct gccgccgttt
     5221 gctttaaaat ctccataatt aagcgccatt agaaactagt cgataaccag aagcaataaa
     5281 aaatcaaatc ggatttcact atataatctc actttatcta agatgaatcc gatggaagca
     5341 tcctgttttc tctcaatttt tttatctaaa acccagcgtt cgatgcttct ttgagcgaac
     5401 gatcaaaaat aagtgccttc ccatcaaaaa aatattctca acataaaaaa ctttgtgtaa
     5461 tacttgtaac gctacatgga gattaactca atctagaggg tattaataat gatccacagt
     5521 agcgtaaagc gttggggaaa ttcaccggcg gtgcggattc cggctacgtt aatgcaggcg
     5581 ctcaatctga atattgatga tgaagtgaag attgacctgg tggatggcaa attaattatt
     5641 gagccagtgc gtaaagagcc cgtatttacg cttgctgaac tggtcaacga catcacgccg
     5701 gaaaacctcc acgagaatat cgactgggga gagccgaaag ataaggaagt ctggtaagag
     5761 ctctaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5821 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5881 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5941 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6001 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6061 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6121 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6181 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6241 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6301 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6361 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6421 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6481 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6541 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6601 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6661 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6721 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6781 gatccttagc gcttctaggg acctctttag ctccttggaa gctgtcagta gtatacctaa
     6841 taatttatct acattccctt tagtaacgtg taactttcca aatttacaaa agcgactcat
     6901 agaattattt cctcccgtta aataatagat aactattaaa aatagacaat acttgctcat
     6961 aagtaacggt acttaaattg tttactttgg cgtgtttcat tgcttgatga aactgatttt
     7021 tagtaaacag ttgacgatat tctcgattga cccattttga aacaaagtac gtatatagct
     7081 tccaatattt atctggaaca tctgtggtat ggcgggtaag ttttattaag acactgttta
     7141 cttttggttt aggatgaaag cattccgctg gcagcttaag caattgctga atcgagactt
     7201 gagtgtgcaa gagcaaccct agtgttcggt gaatatccaa ggtacgcttg tagaatcctt
     7261 cttcaacaat cagatagatg tcagacgcat ggctttcaaa aaccactttt ttaataattt
     7321 gtgtgcttaa atggtaagga atactcccaa caattttata cctctgtttg ttagggaatt
     7381 gaaactgtag aatatcttgg tgaattaaag tgacacgagt attcagtttt aatttttctg
     7441 acgataagtt gaatagatga ctgtctaatt caatagacgt tacctgttta cttattttag
     7501 ccagtttcgt cgttaaatgc cctttacctg ttccaatttc gtaaacggta tcggtttctt
     7561 ttaaattcaa ttgttttatt atttggttga gtactttttc actcgttaaa aagttttgag
     7621 aatattttat atttttgttc atgtaatcac tccttcttaa ttacaaattt ttagcatcta
     7681 atttaacttc aattcctatt atacaaaatt ttaagatact gcactatcaa cacactctta
     7741 agtttgcttc taagtcttat ttccataact tcttttacgt ttccgccatt ctttgctgtt
     7801 tcgattttta tgatatggtg caagtcagca cgaacacgaa ccgtcttatc tcccattata
     7861 tctttttttg cactgattgg tgtatcattt cgtttttctt tttgtgctag aggatcaatt
     7921 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atga
//
