LOCUS       sequence-129049-        2484 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA for dpy-10 conversion using NGCG PAM and VRER Cas9 
            mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2484)
  AUTHORS   Bell RT, Fu BX, Fire AZ
  TITLE     Cas9 Variants Expand the Target Repertoire in Caenorhabditis 
            elegans.
  JOURNAL   Genetics. 2015 Dec 17. pii: genetics.115.185041.
  PUBMED    26680661
REFERENCE   2  (bases 1 to 2484)
  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: "Genetics. 
            2015 Dec 17. pii: genetics.115.185041."
COMMENT     Sequence Label: pRB1084
FEATURES             Location/Qualifiers
     source          1..2484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..22
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6..22
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        197..546
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans R07E5.16 U6 snRNA gene 
                     (Dickinson et al., 2013)"
     misc_RNA        567..642
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(785..801)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(785..801)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      849..880
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        881..983
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             984..1799
                     /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="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1057..1076)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1823..1850
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(1862..2449)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1942..1961)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cccgtgtaaa acgacggcca gtttatctag tcagcttgat tctagctgat cgtggaccgg
       61 aaggtgagcc agtgagttga ttgcagtcca gttacgctgg agtctgaggc tcgtcctgaa
      121 tgatatgcga ccgccggagg gttgcgtttg agacgggcga cagatccagt cgcgctgctc
      181 tcgtcgatcc aagcttcaaa aaaaactagc aataaaggaa taaaaaactg tacaccttaa
      241 aggcgcacac tctgttttgc aaattttatt tttagttgtg aattttctgc tgagacttga
      301 aaatagcaac tttagtacta ctataatttg tcaacctttt caaaaaaagc atgcaatttt
      361 tgagaaactc ttataaaagc tattattaaa aaaacacctt ttttccaaaa ttattccaca
      421 aaaaatatgt tatgaaatgc ctacaccctc tcacacacac tctttatact actctgtcaa
      481 actcacgaga tgtctgccgc ctcttgtgtt gcccctatat aaacacctcc tattgcgaga
      541 tgtcttgcgc taccataggc accacggttt tagagctaga aatagcaagt taaaataagg
      601 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttgtg aaatttgcta
      661 gcggtgcgag cggatcgagc agtgtcgatc actactggac cgcgagctgt gctgcgaccc
      721 gtgatcttac ggcattatac gtatgatcgg tccacgatca gctagattat ctagtcagct
      781 tgatgtcata gctgtttcct gaggctcaat actgaccatt taaatcatac ctgacctcca
      841 tagcagaaag tcaaaagcct ccgaccggag gcttttgact tgatcggcac gtaagaggtt
      901 ccaactttca ccataatgaa ataagatcac taccgggcgt attttttgag ttatcgagat
      961 tttcaggagc taaggaagct aaaatgagcc atattcaacg ggaaacgtct tgcttgaagc
     1021 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     1081 tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt
     1141 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcaggctaa
     1201 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     1261 atgcatggtt actcaccact gcgatcccag ggaaaacagc attccaggta ttagaagaat
     1321 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     1381 cgattcctgt ttgtaattgt ccttttaacg gcgatcgcgt atttcgtctc gctcaggcgc
     1441 aatcacgaat gaataacggt ttggttggtg cgagtgattt tgatgacgag cgtaatggct
     1501 ggcctgttga acaagtctgg aaagaaatgc ataaactctt gccattctca ccggattcag
     1561 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     1621 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     1681 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     1741 ttgataatcc tgatatgaat aaattgcagt ttcacttgat gctcgatgag tttttctaat
     1801 gaggacctaa atgtaatcac ctggctcacc ttcgggtggg cctttctgcg ttgctggcgt
     1861 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgatgctca agtcagaggt
     1921 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     1981 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2041 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2101 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2161 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2221 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2281 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2341 cctcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2401 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2461 gattttctac cgaagaaagg ccca
//
