LOCUS       sequence-364092-        3821 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Directed evolution of Cas9 adenine base editors with altered PAM 
            compatibility.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3821)
  AUTHORS   Huang TP, Heins ZJ, Miller SM, Wong BG, Balivada PA, Wang T, Khalil 
            AS, Liu DR
  TITLE     High-throughput continuous evolution of compact Cas9 variants 
            targeting single-nucleotide-pyrimidine PAMs.
  JOURNAL   Nat Biotechnol. 2022 Sep 8. pii: 10.1038/s41587-022-01410-2. doi: 
            10.1038/s41587-022-01410-2.
  PUBMED    36076084
REFERENCE   2  (bases 1 to 3821)
  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"; journalName: "Nat 
            Biotechnol. 2022 Sep 8. pii: 10.1038/s41587-022-01410-2. doi: 
            10.1038/s41587-022-01410-2."
COMMENT     Sequence Label: pTPH412
FEATURES             Location/Qualifiers
     source          1..3821
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(3..953)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      1001..1223
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(2898..3713)
                     /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(2898..3713)
                     /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     3621..3640
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3705..3724
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 gttcagatcc ttccgtattt agccagtatg ttctctagtg tggttcgttg tttttgcgtg
       61 agccatgaga acgaaccatt gagatcatgc ttactttgca tgtcactcaa aaattttgcc
      121 tcaaaactgg tgagctgaat ttttgcagtt aaagcatcgt gtagtgtttt tcttagtccg
      181 ttacgtaggt aggaatctga tgtaatggtt gttggtattt tgtcaccatt catttttatc
      241 tggttgttct caagttcggt tacgagatcc atttgtctat ctagttcaac ttggaaaatc
      301 aacgtatcag tcgggcggcc tcgcttatca accaccaatt tcatattgct gtaagtgttt
      361 aaatctttac ttattggttt caaaacccat tggttaagcc ttttaaactc atggtagtta
      421 ttttcaagca ttaacatgaa cttaaattca tcaaggctaa tctctatatt tgccttgtga
      481 gttttctttt gtgttagttc ttttaataac cactcataaa tcctcataga gtatttgttt
      541 tcaaaagact taacatgttc cagattatat tttatgaatt tttttaactg gaaaagataa
      601 ggcaatatct cttcactaaa aactaattct aatttttcgc ttgagaactt ggcatagttt
      661 gtccactgga aaatctcaaa gcctttaacc aaaggattcc tgatttccac agttctcgtc
      721 atcagctctc tggttgcttt agctaataca ccataagcat tttccctact gatgttcatc
      781 atctgagcgt attggttata agtgaacgat accgtccgtt ctttccttgt agggttttca
      841 atcgtggggt tgagtagtgc cacacagcat aaaattagct tggtttcatg ctccgttaag
      901 tcatagcgac taatcgctag ttcatttgct ttgaaaacaa ctaattcaga catacatctc
      961 aattggtcta ggtgatttta atcactatac caattgagat gggctagtca atgataatta
     1021 ctagtccttt tcctttgagt tgtgggtatc tgtaaattct gctagacctt tgctggaaaa
     1081 cttgtaaatt ctgctagacc ctctgtaaat tccgctagac ctttgtgtgt tttttttgtt
     1141 tatattcaag tggttataat ttatagaata aagaaagaat aaaaaaagat aaaaagaata
     1201 gatcccagcc ctgtgtataa ctcactactt tagtcagttc cgcagtatta caaaaggatg
     1261 tcgcaaacgc tgtttgctcc tctacaaaac agaccttaaa accctaaagg cttaagtagc
     1321 accctcgcaa gctcggacaa atcgctgaat attccttttg tctccgacca tcaggcacct
     1381 gagtcgctgt ctttttcgtg acattcagtt cgctgcgctc acggctctgg cagtgaatgg
     1441 gggtaaatgg cactacaggc gccttttatg gattcatgca aggaaactac ccataataca
     1501 agaaaagccc gtcacgggct tctcagggcg ttttatggcg ggtctgctat gtggtgctat
     1561 ctgacttttt gctgttcagc agttcctgcc ctctgatttt ccagtctgac cacttcggat
     1621 tatcccgtga caggtcattc agactggcta atgcacccag taaggcagcg gtatcatcaa
     1681 caggcttacc cgtcttactg tcaagaggac atccggtcaa ataaaacgaa aggctcagtc
     1741 gaaagactgg gcctttcgtt ttgctgagga gacttaggga ccctgatgaa attcgccact
     1801 tgttagtgta attcgctaac tcatcctggc atgttgctgt tgattcttca atcagatctc
     1861 tataaatcaa aaagataaaa aattggcacg caaattgtat taacagttca gcaggacaat
     1921 cctgaacgca gaaatcaaga ggacaacatt acctgcaggt gcagtaaagg aaaaaaaaaa
     1981 tgtctgaggt ggagttttcc cacgagtact ggatgagaca tgccctgacc ctggccaaga
     2041 gggcacggga tgagggggag gtgcctgtgg gagccgtgct ggtgctgaac aatagagtga
     2101 tcggcgaggg ctggaacaga gccatcggcc tgcacgaccc aacagcccat gccgaaatta
     2161 tggccctgag acagggcggc ctggtcatgc agaactacag actgattgac gccaccctgt
     2221 acgtgacatt cgagccttgc gtgatgtgcg ccggcgccat gatccactct aggatcggcc
     2281 gcgtggtgtt tggcgtgaga aactcaaaaa gaggcgccgc aggctccctg atgaacgtgc
     2341 tgaactaccc cggcatgaat caccgcgtcg aaattaccga gggaatcctg gcagatgaat
     2401 gtgccgccct gctgtgcgat ttctatcgga tgcctagaca ggtgttcaat gctcagaaga
     2461 aggcccagag ctccatcaac aaggaatctg gctctgtatc ttctgaacag ttggcgcaat
     2521 tccgctctct ggacagccag ctgaaccgct gcctgagcct ggataccatg gtggtgacca
     2581 acggcaaagc gattgaaatt cgcgatgtga aagtgggcga ttggctggaa agcgaatgcg
     2641 gcccggtgca ggtgaccgaa gtgctgccga ttattaaaca gccggtgttt gaaattgtgc
     2701 tgaaaagcgg caaaaaaatt cgcgtgagcg cgaaccataa atttccgacc aaagatggcc
     2761 tgaaaaccat taacagcggc ctgaaagtgg gcgattttct gcgcagccgc gcgaaataac
     2821 ttaattaacg gcactcctca gcaaatataa tgaccctctt gataacccaa gagggcattt
     2881 tttaatgccc atggcgttta gaaaaactca tcgagcatca aatgaaactg caatttattc
     2941 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     3001 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     3061 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     3121 tcaccatgag tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag
     3181 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     3241 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     3301 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     3361 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     3421 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     3481 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     3541 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     3601 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     3661 ttggaattta atcgcggcct cgagcaagac gtttcccgtt gaatatggct cataacaccc
     3721 cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct
     3781 tgtgcaatgt aacatcagag attttgaagg ccaaataggc c
//
