LOCUS       sequence-296569-        2350 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a pegRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2350)
  AUTHORS   Schmid-Burgk JL, Gao L, Li D, Gardner Z, Strecker J, Lash B, Zhang F
  TITLE     Highly Parallel Profiling of Cas9 Variant Specificity.
  JOURNAL   Mol Cell. 2020 Mar 17. pii: S1097-2765(20)30143-X. doi: 
            10.1016/j.molcel.2020.02.023.
  PUBMED    32187529
REFERENCE   2  (bases 1 to 2350)
  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: "Mol Cell. 
            2020 Mar 17. pii: S1097-2765(20)30143-X. doi: 
            10.1016/j.molcel.2020.02.023."
COMMENT     Sequence Label: pU6-pegRNA-HEK3+24
FEATURES             Location/Qualifiers
     source          1..2350
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(380..403)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     rep_origin      complement(520..1108)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(600..619)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1279..2070,2071..2139))
                     /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     1902..1921
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2140..2244)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gcccagactg agcacgtgag ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgctggag gaagcagggc
      361 ttcctttcct ctgccatcac ttatcgtcgt catccttgta atccgtgctc agtctgtttt
      421 tttaagcttg ggccgctcga ggtacctctc tacatatgac atgtgagcaa aaggccagca
      481 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      541 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      601 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
      661 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
      721 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
      781 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
      841 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
      901 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
      961 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     1021 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     1081 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     1141 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     1201 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1261 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     1321 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     1381 gggcttacca tctggcccca gtgctgcaat gataccgcga gatccacgct caccggctcc
     1441 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     1501 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     1561 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     1621 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     1681 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     1741 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     1801 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     1861 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     1921 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     1981 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     2041 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     2101 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     2161 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2221 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgctag
     2281 ctgtacaaaa aagcaggctt taaaggaacc aattcagtcg actggatccg gtaccaaggt
     2341 cgggcaggaa
//
