LOCUS       sequence-296570-        2362 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 2362)
  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 2362)
  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-DNMT1+38
FEATURES             Location/Qualifiers
     source          1..2362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        71..319
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     71..91
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     242..261
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        340..415
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    446..483
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     rep_origin      complement(602..1190)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(682..701)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1361..2152,2153..2221))
                     /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     1984..2003
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2222..2326)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgtacaaaa aagcaggctt taaaggaacc aattcagtcg actggatccg gtaccaaggt
       61 cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
      121 tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
      181 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
      241 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
      301 gtggaaagga cgaaacaccg gtgccagaaa caggggtgag ttttagagct agaaatagca
      361 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcctgct
      421 aaggactagt tctgccctcc agtcaggctt gtcgacgacg gcggtctccg tcgtcaggat
      481 catcccctgt ttctggcatt tttttaagct tgggccgctc gaggtacctc tctacatatg
      541 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      601 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      661 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      721 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      781 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      841 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      901 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      961 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     1021 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     1081 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     1141 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     1201 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     1261 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     1321 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     1381 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1441 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1501 gagatccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1561 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1621 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1681 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1741 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1801 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1861 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1921 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1981 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     2041 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     2101 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     2161 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     2221 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     2281 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     2341 aagtgccacc tgacgtcgct ag
//
