LOCUS       sequence-320075-        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 GAPDH
FEATURES             Location/Qualifiers
     source          1..2279
                     /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"
     rep_origin      complement(449..1037)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(529..548)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1208..1999,2000..2068))
                     /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     1831..1850
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2069..2173)
                     /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 gtccaggggt cttactcctg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttaagcttgg
      361 gccgctcgag gtacctctct acatatgaca tgtgagcaaa aggccagcaa aaggccagga
      421 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
      481 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
      541 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
      601 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
      661 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
      721 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
      781 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
      841 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
      901 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
      961 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     1021 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     1081 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     1141 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     1201 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     1261 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     1321 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     1381 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     1441 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     1501 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     1561 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     1621 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     1681 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     1741 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     1801 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     1861 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     1921 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     1981 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     2041 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     2101 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     2161 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgctagc tgtacaaaaa
     2221 agcaggcttt aaaggaacca attcagtcga ctggatccgg taccaaggtc gggcaggaa
//
