LOCUS       sequence-449949-        2280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human gRNA expression vector targeting the Y93H mutation of 
            non-fluorescent EGFP in plasmid pCBE-dGFP; for use with CBEs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2280)
  AUTHORS   Osgood NRB, Zawalick NM, Sawyer CB, Cowan QT, Gu S, Mawson SJ, 
            Ranzau BL, Li L, Gymrek M, Goren A, Komor AC
  TITLE     Genome editing with programmable base editors in human cells
  JOURNAL   Methods in Enzymology, February 5, 2025
REFERENCE   2  (bases 1 to 2280)
  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: "Methods in 
            Enzymology, February 5, 2025"
COMMENT     Sequence Label: pCBE-dGFP-gRNA
FEATURES             Location/Qualifiers
     source          1..2280
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(268..372)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        479..727
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     479..499
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     650..669
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        749..824
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(928..1516)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1008..1027)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1687..2280,1..198,199..267))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
       61 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
      121 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
      181 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
      241 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
      301 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
      361 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgctagct gtacaaaaaa
      421 gcaggcttta aaggaaccaa ttcagtcgac tggatccggt accaaggtcg ggcaggaaga
      481 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
      541 aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
      601 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
      661 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
      721 aaacaccgag gtcacgtaca ggagcgcagt tttagagcta gaaatagcaa gttaaaataa
      781 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt taagcttggg
      841 ccgctcgagg tacctctcta catatgacat gtgagcaaaa ggccagcaaa aggccaggaa
      901 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
      961 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     1021 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     1081 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     1141 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     1201 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     1261 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     1321 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     1381 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     1441 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     1501 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     1561 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     1621 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     1681 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     1741 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     1801 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     1861 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     1921 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     1981 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     2041 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     2101 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     2161 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     2221 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
//
