LOCUS       sequence-149708-        7572 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacillus subtilis sgRNA expression vector; integrates into amyE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7572)
  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 7572)
  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: pJMP2
FEATURES             Location/Qualifiers
     source          1..7572
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        745..820
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             841..870
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             886..903
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(959..976)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1129..1175
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2363..2382)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      complement(5743..6331)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5823..5842)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6502..7293,7294..7362))
                     /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     7125..7144
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7363..7467)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7535..7553
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga
       61 gggatttttg actccgaagt aagtcttcaa aaaatcaaat aaggagtgtc aagaatgttt
      121 gcaaaacgat tcaaaacctc tttactgccg ttattcgctg gatttttatt gctgtttcat
      181 ttggttctgg caggaccggc ggctgcgagt gctgaaacgg cgaacaaatc gaatgagctt
      241 acagcaccgt cgatcaaaag cggaaccatt cttcatgcat ggaattggtc gttcaatacg
      301 ttaaaacaca atatgaagga tattcatgat gcaggatata cagccattca gacatctccg
      361 attaaccaag taaaggaagg gaatcaagga gataaaagca tgtcgaactg gtactggctg
      421 tatcagccga catcgtatca aattggcaac cgttacttag gtactgaaca agaatttaaa
      481 gaaatgtgtg cagccgctga agaatatggc ataaaggtca ttgttgacgc ggtcatcaat
      541 cataccacca gtgattatgc cgcgatttcc aatgaggtta agagtattcc aaactggaca
      601 catggaaaca cacaaattaa aaactggtct gatcggatcc tagaagctta tcgaattcct
      661 tattaacgtt gatataattt aaattttatt tgacaaaaat gggctcgtgt tgtacaataa
      721 atgtaacttt cagtttagcg gtctgtttta gagctagaaa tagcaagtta aaataaggct
      781 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttgaa gcttgggccc
      841 gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat
      901 cattgagttt aaacggtctc cagcttggct gttttggcgg atgagagaag attttcagcc
      961 tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca
     1021 gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg
     1081 atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga
     1141 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaactggatc
     1201 cttactcgag tctagactgc aggcttcctc gctcactgac tcgaattctc atgtttgaca
     1261 gcttatcatc ggcaatagtt acccttatta tcaagataag aaagaaaagg atttttcgct
     1321 acgctcaaat cctttaaaaa aacacaaaag accacatttt ttaatgtggt ctttattctt
     1381 caactaaagc acccattagt tcaacaaacg aaaattggat aaagtgggat atttttaaaa
     1441 tatatattta tgttacagta atattgactt ttaaaaaagg attgattcta atgaagaaag
     1501 cagacaagta agcctcctaa attcacttta gataaaaatt taggaggcat atcaaatgaa
     1561 ctttaataaa attgatttag acaattggaa gagaaaagag atatttaatc attatttgaa
     1621 ccaacaaacg acttttagta taaccacaga aattgatatt agtgttttat accgaaacat
     1681 aaaacaagaa ggatataaat tttaccctgc atttattttc ttagtgacaa gggtgataaa
     1741 ctcaaataca gcttttagaa ctggttacaa tagcgacgga gagttaggtt attgggataa
     1801 gttagagcca ctttatacaa tttttgatgg tgtatctaaa acattctctg gtatttggac
     1861 tcctgtaaag aatgacttca aagagtttta tgatttatac ctttctgatg tagagaaata
     1921 taatggttcg gggaaattgt ttcccaaaac acctatacct gaaaatgctt tttctctttc
     1981 tattattcca tggacttcat ttactgggtt taacttaaat atcaataata atagtaatta
     2041 ccttctaccc attattacag caggaaaatt cattaataaa ggtaattcaa tatatttacc
     2101 gctatcttta caggtacatc attctgtttg tgatggttat catgcaggat tgtttatgaa
     2161 ctctattcag gaattgtcag ataggcctaa tgactggctt ttataatatg agataatgcc
     2221 gactgtactt tttacagtcg gttttctaat gtcactaacc tgccccgtta gttgaagaag
     2281 gtttttatat tacagctcca gatcctctac gccggacgca tcgtggccgg catcaccggc
     2341 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
     2401 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
     2461 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac
     2521 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
     2581 catggatgag cgatgatgat atccgtttag gctgggcggt gatagcttct cgttcaggca
     2641 gtacgcctct tttcttttcc agacctgagg gaggcggaaa tggtgtgagg ttcccgggga
     2701 aaagccaaat aggcgatcgc gggagtgctt tatttgaaga tcaggctatc actgcggtca
     2761 atagatttca caatgtgatg gctggacagc ctgaggaact ctcgaacccg aatggaaaca
     2821 accagatatt tatgaatcag cgcggctcac atggcgttgt gctggcaaat gcaggttcat
     2881 cctctgtctc tatcaatacg gcaacaaaat tgcctgatgg caggtatgac aataaagctg
     2941 gagcgggttc atttcaagtg aacgatggta aactgacagg cacgatcaat gccaggtctg
     3001 tagctgtgct ttatcctgat gatattgcaa aagcgcctca tgttttcctt gagaattaca
     3061 aaacaggtgt aacacattct ttcaatgatc aactgacgat taccttgcgt gcagatgcga
     3121 atacaacaaa agccgtttat caaatcaata atggaccaga cgacaggcgt ttaaggatgg
     3181 agatcaattc acaatcggaa aaggagatcc aatttggcaa aacatacacc atcatgttaa
     3241 aaggaacgaa cagtgatggt gtaacgagga ccgagaaata cagttttgtt aaaagagatc
     3301 cagcgtcggc caaaaccatc ggctatcaaa atccgaatca ttggagccag gtaaatgctt
     3361 atatctataa acatgatggg agccgagtaa ttgaattgac cggatcttgg cctggaaaac
     3421 caatgactaa aaatgcagac ggaatttaca cgctgacgct gcctgcggac acggatacaa
     3481 ccaacgcaaa agtgattttt aataatggca gcgcccaagt gcccggtcag aatcagcctg
     3541 gctttgatta cgtgctaaat ggtttatata atgactcggg cttaagcggt tctcttcccc
     3601 attgagggca aggctagacg ggacttaccg aaagaaacca tcaatgatgg tttctttttt
     3661 gttcataaat cagacaaaac ttttctcttg caaaagtttg tgaagtgttg cacaatataa
     3721 atgtgaaata cttcacaaac aaaaagacat caaagagaaa cataccctgc aaggatgctg
     3781 atattgtctg catttgcgcc ggagcaaacc aaaaacctgg tgagacacgc cttgaattag
     3841 tagaaaagaa cttgaagatt ttcaaaggca tcgttagtga agtcatggcg agcggatttg
     3901 acggcatttt cttagtcgcg acgcgaggct ggatggcctt ccccattatg attcttctcg
     3961 cttccggcgg catcgggatg cccgcgttgc aggccatgct gtccaggcag gtagatgacg
     4021 accatcaggg acagcttcaa ggatcgctcg cggctcttac cagcctaact tcgatcactg
     4081 gaccgctgat cgtcacggcg atttatgccg cctcggcgag cacatggaac gggttggcat
     4141 ggattgtagg cgccgcccta taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga
     4201 gccgggccac ctactgaagt ggatttcttt aagagctcct ttaacttcct caccagtagt
     4261 tgtatcggta ccataagtag aagcagcaac ccaagtagct ttaccagcat ccggttcaac
     4321 cagcatagta agaatcttac tggacatcgg cagttcttcg aacagtgcgc caactaccag
     4381 ctctttctgc agttcattca gggcaccgga gaacctgcgt gcaatccatc ttgttcaatc
     4441 atgcgaaacg atcctcatcc tgtctcttga tccatggatt acgcgttaac ccgggcccgc
     4501 ggatgcatat gatcagatct taaggcctag gtctagagga tcgatctgta taataaagaa
     4561 taattattaa tctgtagaca aattgtgaaa ggatgtactt aaacgctaac ggtcagcttt
     4621 attgaacagt aatttaagta tatgtccaat ctagggtaag taaattgagt atcaatataa
     4681 actttatatg aacataatca acgaggtgaa atcatgagca atttgattaa cggaaaaata
     4741 ccaaatcaag cgattcaaac attaaaaatc gtaaaagatt tatttggaag ttcaatagtt
     4801 ggagtatatc tatttggttc agcagtaaat ggtggtttac gcattaacag cgatgtagat
     4861 gttctagtcg tcgtgaatca tagtttacct caattaactc gaaaaaaact aacagaaaga
     4921 ctaatgacta tatcaggaaa gattggaaat acggattctg ttagaccact tgaagttacg
     4981 gttataaata ggagtgaagt tgtcccttgg caatatcctc caaaaagaga atttatatac
     5041 ggtgagtggc tcaggggtga atttgagaat ggacaaattc aggaaccaag ctatgatcct
     5101 gatttggcta ttgttttagc acaagcaaga aagaatagta tttctctatt tggtcctgat
     5161 tcttcaagta tacttgtctc cgtacctttg acagatattc gaagagcaat taaggattct
     5221 ttgccagaac taattgaggg gataaaaggt gatgagcgta atgtaatttt aaccctagct
     5281 cgaatgtggc aaacagtgac tactggtgaa attacctcga aagatgtcgc tgcagaatgg
     5341 gctatacctc ttttacctaa agagcatgta actttactgg atatagctag aaaaggctat
     5401 cggggagagt gtgatgataa gtgggaagga ctatattcaa aggtgaaagc actcgttaag
     5461 tatatgaaaa attctataga aacttctctc aattaggcta attttattgc aataacaggt
     5521 gcttactttt ctggagttct ttagcaaatt tttttattag ctgaacttag tattagtggc
     5581 catactcctc caatccaaag ctatttagaa agattactat atcctcaaac aggcggtaac
     5641 cggcctcttc atcgggaatg cgcgcgacct tcagcatcgc cggcatgtcc ccctggcgga
     5701 cgggaagtat ccagctcgag gtcgggccgc gttgctggcg tttttccata ggctccgccc
     5761 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5821 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5881 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5941 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6001 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6061 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6121 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6181 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttga
     6241 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6301 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6361 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6421 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6481 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6541 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6601 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6661 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6721 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6781 gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc
     6841 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6901 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6961 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7021 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7081 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca
     7141 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7201 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7261 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7321 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7381 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7441 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     7501 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     7561 tcttcaagaa tt
//
