LOCUS       sequence-269658-        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"
     protein_bind    388..409
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(424..429,430..447,448..454)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     misc_RNA        480..555
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(668..1006)
                     /codon_start=1
                     /gene="VI"
                     /product="pVI"
                     /label=M13 gene VI
                     /note="minor coat protein"
                     /translation="MPVLLGIPLLLRFLGFLLVTLFGYLLTFLKKGFGKIAIAISLFLA
                     LIIGLNSILVGYLSDISAQLPSDFVQGVQLILPSNALPCFYVILSVKAAIFIFDVKQKI
                     VSYLDWDK"
     promoter        complement(join(1046..1052,1053..1070,1071..1076))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     rep_origin      1262..1807
                     /direction=RIGHT
                     /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     1708..1727
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1937..1956)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             join(1948..2594,1..169)
                     /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             join(1948..2594,1..169)
                     /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     complement(2021..2040)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
       61 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
      121 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag atccgtacgc
      181 catgggcatt aaaaaatgcc ctcttgggtt atcaagaggg tcattatatt tcgcaaaaaa
      241 ccccgcttcg gcggggtttt ttcgcagtgc cgttaattaa gtgcgcccaa tacgcaaacc
      301 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
      361 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
      421 ggctttacac tttatgcttc cggctcgtat gttgtgtggg tcgcaggaca gcttttcctg
      481 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
      541 gcaccgagtc ggtgcttttt ttacagtcag tagggcccta aaaaaaaacc ccgcccctga
      601 cagggcgggg ttttttttgg cgaaaaaacc ccgctgaagc ggggtttttt gcgtacattg
      661 gctccgttta tttatcccaa tccaaataag aaacgatttt ttgtttaacg tcaaaaatga
      721 aaatagcagc ctttacagag agaataacat aaaaacaggg aagcgcatta gacgggagaa
      781 ttaactgaac accctgaaca aagtcagagg gtaattgagc gctaatatca gagagataac
      841 ccacaagaat tgagttaagc ccaataataa gagcaagaaa caatgaaata gcaatagcta
      901 tcttaccgaa gcccttttta agaaaagtaa gcagatagcc gaacaaagtt accagaagga
      961 aaccgaggaa acgcaataat aacggaatac ccaaaagaac tggcattttt ttttcctcct
     1021 tactgcacct gcaggtcggt cgacacaaca tacgagccgg aagcataaag tgtaaagccc
     1081 ggggtgccta atgtaacacc gtgcgtgttg tagggtccct agtcgcagga cagcttttcc
     1141 ttaaacaggt ttctcttctt tccatgcaac gaaaggccca gtctttcgac tgagcctttc
     1201 gttgcatttg cccggatgtc ctctcgtata tcgtatgggg ctccttaata agatgatctt
     1261 cttgagatcg ttttggtctg cgcgtaatat cttgctctga aaacgaaaaa accgccttgc
     1321 agggcggttt ttcgaaggtt ctctgagcta ccaactcttt gaaccgaggt aactggcttg
     1381 gaggagcgca gtcaccaaaa cttgtccttt cagtttagcc ttaaccggcg catgacttca
     1441 agactaactc ctctaaatca attaccagtg gctgctgcca gtggtgcttt tgcatgtctt
     1501 tccgggttgg actcaagacg atagttaccg gataaggcgc agcggtcgga ctgaacgggg
     1561 ggttcgtgca tacagtccag cttggagcga actgcctacc cggaactgag tgtcaggcgt
     1621 ggaatgagac aaacgcggcc ataacagcgg aatgacaccg gtaaaccgaa aggcaggaac
     1681 aggagagcgc acgagggagc cgccaggggg aaacgcctgg tatctttata gtcctgtcgg
     1741 gtttcgccac cactgatttg agcgtcagat ttcgtgatgc ttgtcagggg ggcggagcct
     1801 atggaaaaac ggctttgccg cggccctctc acttccctgt taagtatctt cctggcatct
     1861 tccaggaaat ctccgccccg atccgtaggt ttctcttcat gaacaataaa actgtctgct
     1921 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct
     1981 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     2041 aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag
     2101 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     2161 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     2221 gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa
     2281 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     2341 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     2401 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     2461 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     2521 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     2581 ataggttgta ttga
//
