LOCUS       sequence-269264-        4273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Directed evolution of SpCas10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4273)
  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 4273)
  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: pTW194b63
FEATURES             Location/Qualifiers
     source          1..4273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(130..921)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(922..1013)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1022..1610
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1511..1530
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1764..1781
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1796..1936
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1862..1881)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2022..2044
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2038..2229)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        join(2481..2486,2487..2504,2505..2511)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     misc_RNA        complement(4024..4099)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(4125..4131,4132..4149,4150..4155))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4170..4191
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gaggcggttt gcgtattggg cgcacttaat taacggcact gcgaaaaaac cccgccgaag
       61 cggggttttt tgcgaaatat aatgaccctc ttgataaccc aagagggcat tttttaatgc
      121 ccatggcgtt tatttgccga ctaccttggt gatctcgcct ttcacgtagt ggacaaattc
      181 ttccaactga tctgcgcgcg aggccaagcg atcttcttct tgtccaagat aagcctgtct
      241 agcttcaagt atgacgggct gatactgggc cggcaggcgc tccattgccc agtcggcagc
      301 gacatccttc ggcgcgattt tgccggttac tgcgctgtac caaatgcggg acaacgtaag
      361 cactacattt cgctcatctc cagcccagtc gggcggcgag ttccatagcg ttaaggtttc
      421 atttagcgcc tcaaatagat cctgttcagg aaccggatca aagagttcct ccgccgctgg
      481 acctaccaag gcaacgctat gttctcttgc ttttgtcagc aagatagcca gatcaatgtc
      541 gatcgtggct ggctcgaaga tacctgcaag aatgtcattg cgctgccatt ctccaaattg
      601 cagttcgcgc ttagctggat aacgccacgg aatgatgtcg tcgtgcacaa caatggtgac
      661 ttctacagcg cggagaatct cgctctctcc aggggaagcc gaagtttcca aaaggtcgtt
      721 gatcaaagct cgccgcgttg tttcatcaag ccttacggtc acagtaacca gcaaatcaat
      781 atcactgtgt ggcttcaggc cgccatccac tgcggagccg tacaaatgta cggccagcaa
      841 cgtcggttcg agatggcgct cgatgacgcc aactacctct gatagttgag tcgatacttc
      901 ggcgatcacc gcttccctca tactcttcct ttttcaatat tattgaagca tttatcaggg
      961 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataggc caaataggcc
     1021 gttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     1081 cagcggtggt ttgtttgccg gatcaagagc taccacctct ttttccgaag gtaactggct
     1141 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     1201 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     1261 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     1321 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     1381 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     1441 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     1501 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     1561 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     1621 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     1681 cgttatcccc tgattctgtg gataaccgta ttacagcctt tgagtgagct gataccgctc
     1741 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     1801 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca
     1861 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     1921 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     1981 tcttctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     2041 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     2101 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     2161 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     2221 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     2281 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacaagagg acatccggtc
     2341 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttgctgag gatggaaaga
     2401 agagaaacct gaccgttcgt tttgctgagg agacttaggg accctacaac acgcacggtg
     2461 ttacattagg caccccgggc tttacacttt atgcttccgg ctcgtatgtt gtgtcgaccg
     2521 acctgcaggt gcagtaaaaa aggaaaaaaa tgatcaaaat tgccacgcgt aaatatttag
     2581 gcaaacagaa tgtttatgat atcggtgtcg agcgcgatca taatttcgcg ctgaaaaacg
     2641 gctttatcgc cagcaattgt tttaatgttg ttcctttcta ttctcactcc gctgaaactg
     2701 ttgaaagttg tttagcaaaa ccccatacag aaaattcatt tactaacgtc tggaaagacg
     2761 acaaaacttt agatcgttac gctaactatg agggctgtct gtggaatgct acaggcgttg
     2821 tagtttgtac tggtgacgaa actcagtgtt acggtacatg ggttcctatt gggcttgcta
     2881 tccctgaaaa tgagggtggt ggctctgagg gtggcggttc tgagggtggc ggttctgagg
     2941 gtggcggtac taaacctcct gagtacggtg atacacctat tccgggctat acttatatca
     3001 accctctcga cggcacttat ccgcctggta ctgagcaaaa ccccgctaat cctaatcctt
     3061 ctcttgagga gtctcagcct cttaatactt tcatgtttca gaataatagg ttccgaaata
     3121 ggcagggggc attaactgtt tatacgggca ctgttactca aggcactgac cccgttaaaa
     3181 cttattacca gtacactcct gtatcatcaa aagccatgta tgacgcttac tggaacggta
     3241 aattcagaga ctgcgctttc cattctggct ttaatgagga tccattcgtt tgtgaatatc
     3301 aaggccaatc gtctgacctg cctcaacctc ctgtcaatgc tggcggcggc tctggtggtg
     3361 gttctggtgg cggctctgag ggtggtggct ctgagggtgg cggttctgag ggtggcggct
     3421 ctgagggagg cggttccggt ggtggctctg gttccggtga ttttgattat gaaaagatgg
     3481 caaacgctaa taagggggct atgaccgaaa atgccgatga aaacgcgcta cagtctgacg
     3541 ctaaaggcaa acttgattct gtcgctactg attacggtgc tgctatcgat ggtttcattg
     3601 gtgacgtttc cggccttgct aatggtaatg gtgctactgg tgattttgct ggctctaatt
     3661 cccaaatggc tcaagtcggt gacggtgata attcaccttt aatgaataat ttccgtcaat
     3721 atttaccttc cctccctcaa tcggttgaat gtcgcccttt tgtctttggc gctggtaaac
     3781 cttacgagtt cagtatcgac tgcgataaga tcaacctgtt ccgcggtgtc tttgcgtttc
     3841 ttttatatgt tgccaccttt atgtatgtat tttctacgtt tgctaacata ctgcgtaata
     3901 aggagtctta aacggagcca atgtacgcaa aaaaccccgc ttcagcgggg ttttttcgcc
     3961 aaaaaaaacc ccgccctgtc aggggcgggg ttttttttta gggccctact gactgtaaaa
     4021 aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg
     4081 ctatttctag ctctaaaact tcgttttgct gaggagactc cacacaacat acgagccgga
     4141 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4201 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4261 caacgcgcgg gga
//
