LOCUS       sequence-67681-e        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: pFYF1327 EGFP Site#2
FEATURES             Location/Qualifiers
     source          1..2279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     82..102
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     253..272
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        351..426
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(530..1118)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(610..629)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1289..2080,2081..2149))
                     /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     1912..1931
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2150..2254)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc
       61 ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata
      121 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      181 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      241 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      301 ttatatatct tgtggaaagg acgaaacacc gatgccgttc ttctgcttgt gttttagagc
      361 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
      421 cggtgctttt tttaagcttg ggccgctcga ggtacctctc tacatatgac atgtgagcaa
      481 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
      541 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
      601 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
      661 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
      721 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
      781 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
      841 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
      901 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
      961 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     1021 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     1081 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     1141 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     1201 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1261 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     1321 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     1381 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     1441 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     1501 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     1561 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     1621 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     1681 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     1741 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     1801 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     1861 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     1921 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     1981 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     2041 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     2101 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     2161 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     2221 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacct
//
