LOCUS       sequence-320076-        2279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2279)
  AUTHORS   Sreekanth V, Zhou Q, Kokkonda P, Bermudez-Cabrera HC, Lim D, Law BK,
            Holmes BR, Chaudhary SK, Pergu R, Leger BS, Walker JA, Gifford DK, 
            Sherwood RI, Choudhary A
  TITLE     Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome 
            Editing Outcomes.
  JOURNAL   ACS Cent Sci. 2020 Dec 23;6(12):2228-2237. doi: 
            10.1021/acscentsci.0c00129. Epub 2020 Nov 18.
  PUBMED    33376784
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"; doi: 
            "10.1021/acscentsci.0c00129"; journalName: "ACS Cent Sci"; date: 
            "2020-12-23- 23"; volume: "6"; issue: "12"; pages: "2228-2237"
COMMENT     Sequence Label: pU6 SgRNA RBM20
FEATURES             Location/Qualifiers
     source          1..2279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(524..1315,1316..1384))
                     /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     1147..1166
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1385..1489)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1596..1844
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1596..1616
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1767..1786
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1865..1940
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(join(2044..2279,1..353))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2124..2143)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
       61 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
      121 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
      181 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
      241 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
      301 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
      361 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
      421 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
      481 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
      541 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
      601 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
      661 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
      721 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
      781 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
      841 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
      901 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
      961 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     1021 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     1081 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     1141 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     1201 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     1261 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     1321 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     1381 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     1441 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     1501 gaaaagtgcc acctgacgtc gctagctgta caaaaaagca ggctttaaag gaaccaattc
     1561 agtcgactgg atccggtacc aaggtcgggc aggaagaggg cctatttccc atgattcctt
     1621 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
     1681 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     1741 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     1801 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggtctc gtagtccggt
     1861 gagcgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     1921 aagtggcacc gagtcggtgc tttttttaag cttgggccgc tcgaggtacc tctctacata
     1981 tgacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2041 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2101 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2161 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2221 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttc
//
