LOCUS       sequence-269641-        3820 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Directed evolution of SpCas11.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3820)
  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 3820)
  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: pTW195b63
FEATURES             Location/Qualifiers
     source          1..3820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(22..28,29..46,47..52))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     rep_origin      681..903
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             951..1901
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     primer_bind     complement(1922..1940)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2008..2112
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2113..2181,2182..2973)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2331..2350)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    3185..3206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3221..3226,3227..3244,3245..3251)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     misc_RNA        3277..3352
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gcacctgcag gtcggtcgac acaacatacg agccggaagc ataaagtgta aagcccgggg
       61 tgcctaatgt aacaccgtgc gtgttgtagg gtccctagga gcccaccgag tacctggcgg
      121 tcaggtttct cttctttcca tgcaacgaaa ggcccagtct ttcgactgag cctttcgttg
      181 catttgcccg gatgtcctct tgaccgtacg acgggtaagc ctgttgatga gcccgctgcc
      241 ttactgggtg cattagccag tctgaatgac ctgtcacggg ataagccgaa gtggtcagac
      301 tggaaaagca gagggcagga actgctgaac agcaaaaagt cagatagcac cacatagcag
      361 acccgccata aaacgccctg agaagcccgt gacgggcttt tcttgtatta tgggtagttt
      421 ccttgcatga atccataaaa ggcgcctgta gtgccattta cccccattca ctgccagagc
      481 cgtgagcgca gcgaactgaa tgtcacgaaa aagacagcga ctcaggtgcc tgatggtcgg
      541 agacaaaagg aatattcagc gatttgcccg agcttgcgag ggtgctactt aagcctttag
      601 ggttttaagg tctgttttgt agaggagcaa acagcgtttg cgacatcctt ttgtaatact
      661 gcggaactga ctaaagtagt gagttataca cagggctggg atctattctt tttatctttt
      721 tttattcttt ctttattcta taaattataa ccacttgaat ataaacaaaa aaaacacaca
      781 aaggtctagc ggaatttaca gagggtctag cagaatttac aagttttcca gcaaaggtct
      841 agcagaattt acagataccc acaactcaaa ggaaaaggac tagtaattat cattgactag
      901 cccatctcaa ttggtatagt gattaaaatt acctagacca attgagatgt atgtctgaat
      961 tagttgtttt caaagcaaat gaactagcga ttagtcgcta tgacttaacg gagcatgaaa
     1021 ccaagctaat tttatgctgt gtggcactac tcaaccccac gattgaaaac cctacaagga
     1081 aagaacggac ggtatcgttc acttataacc aatacgctca gatgatgaac atcagtaggg
     1141 aaaatgctta tggtgtatta gctaaagcaa ccagagagct gatgacgaga actgtggaaa
     1201 tcaggaatcc tttggttaaa ggctttgaga ttttccagtg gacaaactat gccaagttct
     1261 caagcgaaaa attagaatta gtttttagtg aagagatatt gccttatctt ttccagttaa
     1321 aaaaattcat aaaatataat ctggaacatg ttaagtcttt tgaaaacaaa tactctatga
     1381 ggatttatga gtggttatta aaagaactaa cacaaaagaa aactcacaag gcaaatatag
     1441 agattagcct tgatgaattt aagttcatgt taatgcttga aaataactac catgagttta
     1501 aaaggcttaa ccaatgggtt ttgaaaccaa taagtaaaga tttaaacact tacagcaata
     1561 tgaaattggt ggttgataag cgaggccgcc cgactgatac gttgattttc caagttgaac
     1621 tagatagaca aatggatctc gtaaccgaac ttgagaacaa ccagataaaa atgaatggtg
     1681 acaaaatacc aacaaccatt acatcagatt cctacctacg taacggacta agaaaaacac
     1741 tacacgatgc tttaactgca aaaattcagc tcaccagttt tgaggcaaaa tttttgagtg
     1801 acatgcaaag taagcatgat ctcaatggtt cgttctcatg gctcacgcaa aaacaacgaa
     1861 ccacactaga gaacatactg gctaaatacg gaaggatctg aacggcctat ttggcctaag
     1921 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     1981 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2041 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2101 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     2161 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     2221 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     2281 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     2341 gcggtattat cccgtattga cgctgggcaa gagcaactcg gtcgccgcat acactattct
     2401 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     2461 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     2521 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     2581 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     2641 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     2701 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     2761 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     2821 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     2881 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     2941 gagataggtg cctcactgat taagcattgg taaacgccat gggcattaaa aaatgccctc
     3001 ttgggttatc aagagggtca ttatatttcg caaaaaaccc cgcttcggcg gggttttttc
     3061 gcagtgccgt taattaagtg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     3121 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     3181 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     3241 ctcgtatgtt gtgtggggag cccaccgagt acctgggttt tagagctaga aatagcaagt
     3301 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttta
     3361 cagtcagtag ggccctaaaa aaaaaccccg cccctgacag ggcggggttt tttttggcga
     3421 aaaaaccccg ctgaagcggg gttttttgcg tacattggct ccgtttaatt cgggagatta
     3481 tccacgcgca tcaggtctaa ttcgcgttca aaaatttcgt cgattgggag catctgacca
     3541 tctacagtca taaatttgtg atcctttgtg gcacggatta aagatccgtc ttccagacaa
     3601 tattcgaaca cttcctgttc accacggtca tgccactggg ccaccggctg ggtgtagata
     3661 ttgccgttgt tatccactga gtagaccgta cattcaatac gtttttcaac aatcttgccg
     3721 attggcagca gaccatattc gacggtcaag atttcggttt cgtagctgag acataaagga
     3781 attgcgaata ataatttttt catttttttt ttcctttact
//
