LOCUS       sequence-149710-        7263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacillus subtilis sgRNA expression vector; integrates into thrC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7263)
  AUTHORS   Peters JM, Colavin A, Shi H, Czarny TL, Larson MH, Wong S, Hawkins 
            JS, Lu CH, Koo BM, Marta E, Shiver AL, Whitehead EH, Weissman JS, 
            Brown ED, Qi LS, Huang KC, Gross CA
  TITLE     A Comprehensive, CRISPR-based Functional Analysis of Essential Genes
            in Bacteria.
  JOURNAL   Cell. 2016 Jun 2;165(6):1493-506. doi: 10.1016/j.cell.2016.05.003. 
            Epub 2016 May 26.
  PUBMED    27238023
REFERENCE   2  (bases 1 to 7263)
  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"; doi: 
            "10.1016/j.cell.2016.05.003"; journalName: "Cell"; date: "2016-06-2-
            2"; volume: "165"; issue: "6"; pages: "1493-506"
COMMENT     Sequence Label: pJMP3
FEATURES             Location/Qualifiers
     source          1..7263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        858..933
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             954..983
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             999..1016
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1072..1089)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1242..1288
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(5434..6022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5514..5533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6193..6984,6985..7053))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6816..6835
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7054..7158)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7226..7244
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aattcatgta aaagatgagg ttggttcatt ctcgaaaatt acatctgtgt tctcagagcg
       61 gggcgtgacg tttgaaaaaa tccttcagct gccaattaaa ggccatgatg agttagctga
      121 aatcgtaatt gtcacacatc atacatcaga agctgatttc agtgatatcc tgcaaaacct
      181 aaatgatttg gaagtcgttc aagaagtcaa aagcacatat cgtgtagaag ggaacggttg
      241 gagctaatgt ggaaaggact tatccatcaa tataaagaat ttttacctgt aacagatcaa
      301 acaccggcgc taactttaca tgaaggaaac acacctctta ttcacctgcc gaagctgtct
      361 gagcagctcg gaattgagct tcatgtcaaa acggaaggcg tcaatcctac gggatcattt
      421 aaagatcgcg gaatggttat ggctgtggca aaggcaaaag aagaaggcaa tgacacgatt
      481 atgtgcgcgt caacaggtaa cacttccgct gctgcggcag catatgcagc ccgtgctaac
      541 atgaaatgca ttgtcatcat cccgaacgga aaaattgcat ttggaaaact cgctcaagct
      601 gtcatgtacg gagccgagat tatcgcaatt gacggaaact ttgacgatgc gcttaaaatt
      661 gtccgttcca tctgtgagaa atcaccgatt gcccttgtca actcagtcaa cccttaccgc
      721 attgaaggcc aaaaaactgc tgccttcgga tcctagaagc ttatcgaatt ccttattaac
      781 gttgatataa tttaaatttt atttgacaaa aatgggctcg tgttgtacaa taaatgtaac
      841 tttcagttta gcggtctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
      901 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaagcttggg cccgaacaaa
      961 aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcatcat catcattgag
     1021 tttaaacggt ctccagcttg gctgttttgg cggatgagag aagattttca gcctgataca
     1081 gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc
     1141 ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag
     1201 tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc
     1261 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaactgg atccttactc
     1321 gagtctagac tgcaggcttc ctcgctcact gactcgaatt cctgcagccc tggcgaatgg
     1381 cgattttcgt tcgtgaatac atgttataat aactataact aataacgtaa cgtgactggc
     1441 aagagatatt tttaaaacaa tgaataggtt tacacttact ttagttttat ggaaatgaaa
     1501 gatcatatca tatataatct agaataaaat taactaaaat aattattatc tagataaaaa
     1561 atttagaagc caatgaaatc tataaataaa ctaaattaag tttatttaat taacaactat
     1621 ggatataaaa taggtactaa tcaaaatagt gaggaggata tatttgaata catacgaaca
     1681 aattaataaa gtgaaaaaaa tacttcggaa acatttaaaa aataacctta ttggtactta
     1741 catgtttgga tcaggagttg agagtggact aaaaccaaat agtgatcttg actttttagt
     1801 cgtcgtatct gaaccattga cagatcaaag taaagaaata cttatacaaa aaattagacc
     1861 tatttcaaaa aaaataggag ataaaagcaa cttacgatat attgaattaa caattattat
     1921 tcagcaagaa atggtaccgt ggaatcatcc tcccaaacaa gaatttattt atggagaatg
     1981 gttacaagag ctttatgaac aaggatacat tcctcagaag gaattaaatt cagatttaac
     2041 cataatgctt taccaagcaa aacgaaaaaa taaaagaata tacggaaatt atgacttaga
     2101 ggaattacta cctgatattc cattttctga tgtgagaaga gccattatgg attcgtcaga
     2161 ggaattaata gataattatc aggatgatga aaccaactct atattaactt tatgccgtat
     2221 gattttaact atggacacgg gtaaaatcat accaaaagat attgcgggaa atgcagtggc
     2281 tgaatcttct ccattagaac atagggagag aattttgtta gcagttcgta gttatcttgg
     2341 agagaatatt gaatggacta atgaaaatgt aaatttaact ataaactatt taaataacag
     2401 attaaaaaaa ttataaaaaa attgaaaaaa tggtggaaac acttttttca atttttttgt
     2461 tttattattt aatatttggg aaatattcat tctaattggt aatcagattt tagaaaacaa
     2521 taaacccttg cataggggga tctcgaggcc ttccgaaaat gcgcggcttt gaagctgagg
     2581 gtgcggcggc aatcgtgcgc aatgaagtga ttgaaaatcc ggaaacaata gcgacagcca
     2641 ttcgtatcgg aaacccggca agctgggaca aagctgtaaa ggcagccgag gaatccaatg
     2701 ggaaaattga cgaagtcact gatgatgaaa tccttcacgc atatcagctg atcgcccgtg
     2761 tagaaggcgt gtttgcagaa ccaggttctt gcgcgtctat cgcaggagtg ctgaaacagg
     2821 tgaaatccgg agaaattccg aaaggcagca aggtcgtagc tgtgttaaca ggaaacggac
     2881 tgaaagatcc gaacacagcg gtcgacattt cagaaatcaa gcctgtcaca ttgccgactg
     2941 atgaagacag catccttgaa tatgtaaaag gagcggcccg tgtatgaacg aagccgacat
     3001 gctgttctct gtcactgttc ccggaagcac agctaaccta ggccccggct ttgattcagt
     3061 cggaatggcg ctcagcagat atttgaagct gaccgtcttt gaaagcgaca aatggtcttt
     3121 tgaggctgaa acagaaacag tcgccggaat taattcgagc tcctttaact tcctcaccag
     3181 tagttgtatc ggtaccataa gtagaagcag caacccaagt agctttacca gcatccggtt
     3241 caaccagcat agtaagaatc ttactggaca tcggcagttc ttcgaacagt gcgccaacta
     3301 ccagctcttt ctgcagttca ttcagggcac cggagaacct gcgtgcaatc catcttgttc
     3361 aatcatgcga aacgatcctc atcctgtctc ttgatccatg gattacgcgt taacccgggc
     3421 ccgcggatgc atatgatcag atcctttaac tctggcaacc ctcaaaattg aatgagacat
     3481 gctacacctc cggataataa atatatataa acgtatatag atttcataaa gtctaacaca
     3541 ctagacttat ttacttcgta attaagtcgt taaaccgtgt gctctacgac caaaactata
     3601 aaacctttaa gaactttctt tttttacaag aaaaaagaaa ttagataaat ctctcatatc
     3661 ttttattcaa taatcgcatc cgattgcagt ataaatttaa cgatcactca tcatgttcat
     3721 atttatcaga gctcgtgcta taattatact aattttataa ggaggaaaaa atatgggcat
     3781 ttttagtatt tttgtaatca gcacagttca ttatcaacca aacaaaaaat aagtggttat
     3841 aatgaatcgt taataagcaa aattcatata accaaattaa agagggttat aatgaacgag
     3901 aaaaatataa aacacagtca aaactttatt acttcaaaac ataatataga taaaataatg
     3961 acaaatataa gattaaatga acatgataat atctttgaaa tcggctcagg aaaaggccat
     4021 tttacccttg aattagtaaa gaggtgtaat ttcgtaactg ccattgaaat agaccataaa
     4081 ttatgcaaaa ctacagaaaa taaacttgtt gatcacgata atttccaagt tttaaacaag
     4141 gatatattgc agtttaaatt tcctaaaaac caatcctata aaatatatgg taatatacct
     4201 tataacataa gtacggatat aatacgcaaa attgtttttg atagtatagc taatgagatt
     4261 tatttaatcg tggaatacgg gtttgctaaa agattattaa atacaaaacg ctcattggca
     4321 ttacttttaa tggcagaagt tgatatttct atattaagta tggttccaag agaatatttt
     4381 catcctaaac ctaaagtgaa tagctcactt atcagattaa gtagaaaaaa atcaagaata
     4441 tcacacaaag ataaacaaaa gtataattat ttcgttatga aatgggttaa caaagaatac
     4501 aagaaaatat ttacaaaaaa tcaatttaac aattccttaa aacatgcagg aattgacgat
     4561 ttaaacaata ttagctttga acaattctta tctcttttca atagctataa attatttaat
     4621 aagtaagtta agggatgcat aaactgcatc ccttaacttg tttttcgtgt gcctattttt
     4681 tgtgaatcga ttatgtcttt tgcgcagtcg gcttaaacca gttttcgctg gtgcgaaaaa
     4741 agagtgtctt gtgacaccta aattcaaaat ctatcggtca gatttatacc gatttgattt
     4801 tatatattct tgaataacat acgccgagtt atcacataaa agcgggaacc aatcatcaaa
     4861 tttaaacttc attgcataat ccattaaact cttaaattct acgattcctt gttcatcaat
     4921 aaactcaatc atttctttaa ttaatttata tctatctgtt gttgttttct ttaataattc
     4981 atcaacatct acaccgccat aaactatcat atcttctttt tgatatttaa atttattagg
     5041 atcttaaggc ctaggtctag agtctttgtt ttgacgccat tagcgtacgt aacaatcctc
     5101 gttaaaggac aaggacctga gcggaagtgt atcgtacagt agacggagta tactagtata
     5161 gtctatagtc cgtggaatta ttatatttat ctccgacgat attctcatca gtgaaatcca
     5221 gctggagttc tttagcaaat ttttttatta gctgaactta gtattagtgg ccatactcct
     5281 ccaatccaaa gctatttaga aagattacta tatcctcaaa caggcggtaa ccggcctctt
     5341 catcgggaat gcgcgcgacc ttcagcatcg ccggcatgtc cccctggcgg acgggaagta
     5401 tccagctcga ggtcgggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5461 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5521 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5581 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5641 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5701 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5761 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5821 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     5881 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg atagctcttg
     5941 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6001 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6061 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6121 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6181 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6241 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6301 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6361 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6421 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6481 tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg
     6541 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6601 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6661 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6721 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6781 gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac
     6841 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6901 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6961 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7021 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7081 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7141 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     7201 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga
     7261 att
//
