LOCUS       sequence-264395-        2594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Directed evolution of SpCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2594)
  AUTHORS   Miller SM, Wang T, Randolph PB, Arbab M, Shen MW, Huang TP, Matuszek
            Z, Newby GA, Rees HA, Liu DR
  TITLE     Continuous evolution of SpCas9 variants compatible with non-G PAMs.
  JOURNAL   Nat Biotechnol. 2020 Apr;38(4):471-481. doi: 
            10.1038/s41587-020-0412-8. Epub 2020 Feb 10.
  PUBMED    32042170
REFERENCE   2  (bases 1 to 2594)
  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: 
            "10.1038/s41587-020-0412-8"; journalName: "Nat Biotechnol"; date: 
            "2020-04"; volume: "38"; issue: "4"; pages: "471-481"
COMMENT     Sequence Label: pTW170b2
FEATURES             Location/Qualifiers
     source          1..2594
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(152..157,158..175,176..182)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             222..560
                     /codon_start=1
                     /gene="VI"
                     /product="pVI"
                     /label=M13 gene VI
                     /note="minor coat protein"
                     /translation="MPVLLGIPLLLRFLGFLLVTLFGYLLTFLKKGFGKIAIAISLFLA
                     LIIGLNSILVGYLSDISAQLPSDFVQGVQLILPSNALPCFYVILSVKAAIFIFDVKQKI
                     VSYLDWDK"
     misc_RNA        complement(673..748)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(774..780,781..798,799..804))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    819..840
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(1059..1874)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1059..1874)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1782..1801
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1866..1885
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      complement(2015..2560)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2095..2114)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgagaggac atccgggcaa atgcaacgaa aggctcagtc gaaagactgg gcctttcgtt
       61 gcatggaaag aagagaaacc tgattagagg cggggtggag ggggtctagg gaccctacaa
      121 cacgcacggt gttacattag gcaccccggg ctttacactt tatgcttccg gctcgtatgt
      181 tgtgtcgacc gacctgcagg tgcagtaagg aggaaaaaaa aatgccagtt cttttgggta
      241 ttccgttatt attgcgtttc ctcggtttcc ttctggtaac tttgttcggc tatctgctta
      301 cttttcttaa aaagggcttc ggtaagatag ctattgctat ttcattgttt cttgctctta
      361 ttattgggct taactcaatt cttgtgggtt atctctctga tattagcgct caattaccct
      421 ctgactttgt tcagggtgtt cagttaattc tcccgtctaa tgcgcttccc tgtttttatg
      481 ttattctctc tgtaaaggct gctattttca tttttgacgt taaacaaaaa atcgtttctt
      541 atttggattg ggataaataa acggagccaa tgtacgcaaa aaaccccgct tcagcggggt
      601 tttttcgcca aaaaaaaccc cgccctgtca ggggcggggt ttttttttag ggccctactg
      661 actgtaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat
      721 tttaacttgc tatttctagc tctaaaacga ggcggggtgg agggggtccc acacaacata
      781 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
      841 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
      901 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgcactta attaacggca
      961 ctgcgaaaaa accccgccga agcggggttt tttgcgaaat ataatgaccc tcttgataac
     1021 ccaagagggc attttttaat gcccatggcg tacggatctt agaaaaactc atcgagcatc
     1081 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     1141 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     1201 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     1261 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     1321 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     1381 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     1441 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     1501 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     1561 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     1621 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     1681 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     1741 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     1801 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     1861 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     1921 catgaagaga aacctacgga tcggggcgga gatttcctgg aagatgccag gaagatactt
     1981 aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca
     2041 agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat
     2101 accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta
     2161 ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc
     2221 gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc
     2281 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca
     2341 ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta
     2401 aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt
     2461 caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt
     2521 ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaagga
     2581 gccccatacg atat
//
