LOCUS       sequence-291764-        2279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  human gRNA expression vector targeting EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2279)
  AUTHORS   Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD
  TITLE     High-frequency off-target mutagenesis induced by CRISPR-Cas 
            nucleases in human cells.
  JOURNAL   Nat Biotechnol. 2013 Jun 23. doi: 10.1038/nbt.2623.
  PUBMED    23792628
REFERENCE   2  (bases 1 to 2279)
  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: "Nat 
            Biotechnol. 2013 Jun 23. doi: 10.1038/nbt.2623."
COMMENT     Sequence Label: pFYF1320 EGFP Site#1
FEATURES             Location/Qualifiers
     source          1..2279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(157..948,949..1017))
                     /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"
     primer_bind     780..799
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1018..1122)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1229..1477
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1229..1249
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1400..1419
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1498..1573
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(1677..2265)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1757..1776)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
       61 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
      121 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
      181 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
      241 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
      301 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
      361 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
      421 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
      481 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
      541 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
      601 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
      661 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
      721 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
      781 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
      841 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
      901 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
      961 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     1021 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     1081 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     1141 gccacctgac gtcgctagct gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac
     1201 tggatccggt accaaggtcg ggcaggaaga gggcctattt cccatgattc cttcatattt
     1261 gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa
     1321 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     1381 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     1441 cttggcttta tatatcttgt ggaaaggacg aaacaccggg cacgggcagc ttgccgggtt
     1501 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     1561 accgagtcgg tgcttttttt aagcttgggc cgctcgaggt acctctctac atatgacatg
     1621 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1681 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1741 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1801 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     1861 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     1921 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     1981 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2041 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2101 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2161 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2221 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgat
//
