LOCUS       sequence-212960-        2279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MLM3636 backbone containing a gRNA that does not bind to any 
            sequence in the human genome..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2279)
  AUTHORS   St Martin A, Salamango D, Serebrenik A, Shaban N, Brown WL, Donati 
            F, Munagala U, Conticello SG, Harris RS
  TITLE     A fluorescent reporter for quantification and enrichment of DNA 
            editing by APOBEC-Cas9 or cleavage by Cas9 in living cells.
  JOURNAL   Nucleic Acids Res. 2018 May 9. pii: 4994269. doi: 
            10.1093/nar/gky332.
  PUBMED    29746667
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: "Nucleic 
            Acids Res. 2018 May 9. pii: 4994269. doi: 10.1093/nar/gky332."
COMMENT     Sequence Label: Non-specific sgRNA
FEATURES             Location/Qualifiers
     source          1..2279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        38..286
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     38..58
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     209..228
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        307..382
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(486..1074)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(566..585)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1245..2036,2037..2105))
                     /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     1868..1887
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2106..2210)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcagtcgact ggatccggta ccaaggtcgg gcaggaagag ggcctatttc ccatgattcc
       61 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
      121 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
      181 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
      241 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgcac taccagagct
      301 aactcagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      361 aaaagtggca ccgagtcggt gcttttttta agcttgggcc gctcgaggta cctctctaca
      421 tatgacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
      481 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      541 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      601 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      661 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      721 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      781 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      841 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
      901 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
      961 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     1021 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     1081 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     1141 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     1201 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     1261 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     1321 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     1381 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     1441 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     1501 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     1561 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     1621 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     1681 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     1741 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     1801 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     1861 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     1921 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     1981 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     2041 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     2101 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     2161 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     2221 cgaaaagtgc cacctgacgt cgctagctgt acaaaaaagc aggctttaaa ggaaccaat
//
