LOCUS       sequence-269639-        5409 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 5409)
  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 5409)
  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: pTW211b5
FEATURES             Location/Qualifiers
     source          1..5409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(2361..3152,3153..3221))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQDQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2984..3003
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3222..3326)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3394..3412
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(3433..4383)
                     /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      4431..4653
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             join(5388..5409,1..251)
                     /codon_start=1
                     /gene="E. coli rpoZ"
                     /product="DNA-directed RNA polymerase subunit omega"
                     /label=RNAP omega
                     /note="promotes RNA polymerase assembly"
                     /translation="MARVTVQDAVEKNGNRFDLVLVAARRARQMQVGGKDPLVPEENDK
                     TTVIALREIEEGLINNQILDVRERQEQQEQEAAELQAVTAIAEGRR"
ORIGIN
        1 acgctgtaga gaaaaatggt aaccgttttg acctggtact ggtcgccgcg cgtcgcgctc
       61 gtcagatgca ggtaggcgga aaggatccgc tggtaccgga agaaaacgat aaaaccactg
      121 taatcgcgct gcgcgaaatc gaagaaggtc tgatcaacaa ccagatcctc gacgttcgcg
      181 aacgccagga acagcaagag caggaagccg ctgaattaca agccgttacc gctattgctg
      241 aaggtcgtcg tgctgcagac aagaagtact ccattgggct cgctatcggc acaaacagcg
      301 tcggctgggc cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg
      361 gcaataccga tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg
      421 gggagacggc cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa
      481 agaatcggat ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact
      541 ctttcttcca taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc
      601 acccaatctt tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat
      661 atcatctgag gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc
      721 tcgcgctggc gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc
      781 cagacaacag cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt
      841 tcgaagagaa cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc
      901 tgtccaaatc ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg
      961 gcctgtttgg taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact
     1021 tcgacctggc cgaagatgcc aagcttcaac tgagcaaaga cacctacgat gatgatctcg
     1081 acaatctgct ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc
     1141 tgtcagacgc cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc
     1201 cgctgagcgc tagtatgatc aagcgctatg atgagcacca ccaagacttg actttgctga
     1261 aggcccttgt cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta
     1321 aaaatggcta cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta
     1381 ttaagcccat cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag
     1441 aagatctgtt gcgcaaacag cgcactttcg acaatggaag catcccccac cagattcacc
     1501 tgggcgaact gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata
     1561 acagggaaaa gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg
     1621 cccggggaaa ttccagattc gcgtggatga ctcgcaaatc agaagagacc atcactccct
     1681 ggaacttcga ggaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga
     1741 ctaactttga taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg
     1801 agtacttcac agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa
     1861 agccagcatt cctgtctgga gagcagaaga aagctatcgt ggacctcctc ttcaagacga
     1921 accggaaagt taccgtgaaa cagctcaaag aagactattt caaaaagatt gaatgtctca
     1981 gctacgaaac cgaaatcttg accgtcgaat atggtctgct gccaatcggc aagattgttg
     2041 aaaaacgtat tgaatgtacg gtctactcag tggataacaa cggcaatatc tacacccagc
     2101 cggtggccca gtggcatgac cgtggtgaac aggaagtgtt cgaatattgt ctggaagacg
     2161 gatctttaat ccgtgccaca aaggatcaca aatttatgac tgtagatggt cagatgctcc
     2221 caatcgacga aatttttgaa cgcgaattag acctgatgcg cgtggataat ctcccgaatt
     2281 aacttaatta acggcactcc tcagcaaata taatgaccct cttgataacc caagagggca
     2341 ttttttaatg cccatggcgt ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     2401 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     2461 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     2521 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     2581 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     2641 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     2701 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     2761 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     2821 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     2881 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     2941 tgtatgcggc gaccgagttg atcttgcccg gcgtcaatac gggataatac cgcgccacat
     3001 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3061 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     3121 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     3181 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     3241 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     3301 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     3361 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gcccttaggc
     3421 caaataggcc gttcagatcc ttccgtattt agccagtatg ttctctagtg tggttcgttg
     3481 tttttgcgtg agccatgaga acgaaccatt gagatcatgc ttactttgca tgtcactcaa
     3541 aaattttgcc tcaaaactgg tgagctgaat ttttgcagtt aaagcatcgt gtagtgtttt
     3601 tcttagtccg ttacgtaggt aggaatctga tgtaatggtt gttggtattt tgtcaccatt
     3661 catttttatc tggttgttct caagttcggt tacgagatcc atttgtctat ctagttcaac
     3721 ttggaaaatc aacgtatcag tcgggcggcc tcgcttatca accaccaatt tcatattgct
     3781 gtaagtgttt aaatctttac ttattggttt caaaacccat tggttaagcc ttttaaactc
     3841 atggtagtta ttttcaagca ttaacatgaa cttaaattca tcaaggctaa tctctatatt
     3901 tgccttgtga gttttctttt gtgttagttc ttttaataac cactcataaa tcctcataga
     3961 gtatttgttt tcaaaagact taacatgttc cagattatat tttatgaatt tttttaactg
     4021 gaaaagataa ggcaatatct cttcactaaa aactaattct aatttttcgc ttgagaactt
     4081 ggcatagttt gtccactgga aaatctcaaa gcctttaacc aaaggattcc tgatttccac
     4141 agttctcgtc atcagctctc tggttgcttt agctaataca ccataagcat tttccctact
     4201 gatgttcatc atctgagcgt attggttata agtgaacgat accgtccgtt ctttccttgt
     4261 agggttttca atcgtggggt tgagtagtgc cacacagcat aaaattagct tggtttcatg
     4321 ctccgttaag tcatagcgac taatcgctag ttcatttgct ttgaaaacaa ctaattcaga
     4381 catacatctc aattggtcta ggtgatttta atcactatac caattgagat gggctagtca
     4441 atgataatta ctagtccttt tcctttgagt tgtgggtatc tgtaaattct gctagacctt
     4501 tgctggaaaa cttgtaaatt ctgctagacc ctctgtaaat tccgctagac ctttgtgtgt
     4561 tttttttgtt tatattcaag tggttataat ttatagaata aagaaagaat aaaaaaagat
     4621 aaaaagaata gatcccagcc ctgtgtataa ctcactactt tagtcagttc cgcagtatta
     4681 caaaaggatg tcgcaaacgc tgtttgctcc tctacaaaac agaccttaaa accctaaagg
     4741 cttaagtagc accctcgcaa gctcgggcaa atcgctgaat attccttttg tctccgacca
     4801 tcaggcacct gagtcgctgt ctttttcgtg acattcagtt cgctgcgctc acggctctgg
     4861 cagtgaatgg gggtaaatgg cactacaggc gccttttatg gattcatgca aggaaactac
     4921 ccataataca agaaaagccc gtcacgggct tctcagggcg ttttatggcg ggtctgctat
     4981 gtggtgctat ctgacttttt gctgttcagc agttcctgcc ctctgatttt ccagtctgac
     5041 cacttcggat tatcccgtga caggtcattc agactggcta atgcacccag taaggcagcg
     5101 gtatcatcaa caggcttacc cgtcttactg tcaagaggac atccggtcaa ataaaacgaa
     5161 aggctcagtc gaaagactgg gcctttcgtt ttgctgagga gacttaggga ccctcacagc
     5221 taacaccacg tcgtccctat ctgctgccct aggtctatga gtggttgctg gataacttta
     5281 cgggcatgca taaggctcgt aggatatatt cagggagacc acaacggttt ccctctacaa
     5341 ataattttgt ttaactttac ctgcaggtgc agtaaaggaa aaaaaaaatg gcacgcgtaa
     5401 ctgttcagg
//
