LOCUS       sequence-72446-e        2236 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for cloning spacer into sgRNA for NmCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2236)
  TITLE     Thomson/Sontheimer Lab NmCas9 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 2236)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pSmart-Nm-sgRNA-BbsI
FEATURES             Location/Qualifiers
     source          1..2236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        42..290
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     42..62
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     213..232
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        341..433
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     terminator      513..544
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        545..647
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             648..1463
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(721..740)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1487..1514
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(1526..2113)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1606..1625)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2135..2164)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
ORIGIN
        1 ccaagctcgg atccgatatc gacgtcaagg tcgggcagga agagggccta tttcccatga
       61 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga
      121 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
      181 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
      241 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gggtcttcat
      301 tccagaagac aagttgtagc tccctttctc atttcggaaa cgaaatgaga accgttgcta
      361 caataaggcc gtctgaaaag atgtgccgca acgctctgcc ccttaaagct tctgctttaa
      421 ggggcatcgt ttatttcggt ttttttgcga cgaattctct agatatcgct caatactgac
      481 catttaaatc atacctgacc tccatagcag aaagtcaaaa gcctccgacc ggaggctttt
      541 gacttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg
      601 gcgtattttt tgagttatcg agattttcag gagctaagga agctaaaatg agccatattc
      661 aacgggaaac gtcttgctcg aggccgcgat taaattccaa catggatgct gatttatatg
      721 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg
      781 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
      841 ttacagatga gatggtcagg ctaaactggc tgacggaatt tatgcctctt ccgaccatca
      901 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc ccagggaaaa
      961 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     1021 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacggcgatc
     1081 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt ggtgcgagtg
     1141 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataagc
     1201 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     1261 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     1321 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     1381 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcact
     1441 tgatgctcga tgagtttttc taatgagggc ccaaatgtaa tcacctggct caccttcggg
     1501 tgggcctttc tgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1561 aaaatcgatg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1621 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1681 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1741 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1801 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1861 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1921 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     1981 tctgcgctct gctgaagcca gttacctcgg aaaaagagtt ggtagctctt gatccggcaa
     2041 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     2101 aaaaggatct caagaagatc ctttgatttt ctaccgaaga aaggcccacc cgtgaaggtg
     2161 agccagtgag ttgattgcag tccagttacg ctggagtctg aggctcgtcc tgaatgatat
     2221 caagcttgaa ttcgtt
//
