LOCUS       sequence-264394-        6309 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 6309)
  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 6309)
  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: pTW199b2
FEATURES             Location/Qualifiers
     source          1..6309
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..792)
                     /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(793..884)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      893..1481
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1382..1401
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1635..1652
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1667..1807
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1733..1752)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1893..1915
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1909..2100)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        join(2352..2357,2358..2375,2376..2382)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             2421..3695
                     /codon_start=1
                     /gene="III"
                     /product="pIII"
                     /label=M13 gene III
                     /note="minor coat protein"
                     /translation="MKKLLFAIPLVVPFYSHSAETVESCLAKPHTENSFTNVWKDDKTL
                     DRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGG
                     TKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQ
                     GALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQ
                     GQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKM
                     ANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGS
                     NSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVF
                     AFLLYVATFMYVFSTFANILRNKES"
     CDS             3695..4774
                     /codon_start=1
                     /gene="luxA"
                     /product="LuxA luciferase subunit"
                     /label=LuxA
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="MKFGNFLLTYQPPQFSQTEVMKRLVKLGRISEECGFDTVWLLEHH
                     FTEFGLLGNPYVAAAYLLGATKKLNVGTAAIVLPTAHPVRQLEDVNLLDQMSKGRFRFG
                     ICRGLYNKDFRVFGTDMNNSRALAECWYGLIKNGMTEGYMEADNEHIKFHKVKVNPAAY
                     SRGGAPVYVVAESASTTEWAAQFGLPMILSWIINTNEKKAQLELYNEVAQEYGHDIHNI
                     DHCLSYITSVDHDSIKAKEICRKFLGHWYDSYVNATTIFDDSDQTRGYDFNKGQWRDFV
                     LKGHKDTNRRIDYSYEINPVGTPQECIDIIQKDIDATGISNICCGFEANGTVDEIIASM
                     KLFQSDVMPFLKEKQRSLLY"
     CDS             4838..5818
                     /codon_start=1
                     /gene="luxB"
                     /product="LuxB luciferase subunit"
                     /label=LuxB
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="KFGLFFLNFINSTTVQEQSIVRMQEITEYVDKLNFEQILVYENHF
                     SDNGVVGAPLTVSGFLLGLTEKIKIGSLNHIITTHHPVRIAEEACLLDQLSEGRFILGF
                     SDCEKKDEMHFFNRPVEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAYT
                     PGGPRKYVTATSHHIVEWAAKKGIPLIFKWDDSNDVRYEYAERYKAVADKYDVDLSEID
                     HQLMILVNYNEDSNKAKQETRAFISDYVLEMHPNENFENKLEEIIAENAVGNYTECITA
                     AKLAIEKCGAKSVLLSFEPMNDLMSQKNVINIVDDNIKKYHTEYT"
     misc_RNA        complement(5931..6006)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(6032..6038,6039..6056,6057..6062))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6077..6098
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ttatttgccg actaccttgg tgatctcgcc tttcacgtag tggacaaatt cttccaactg
       61 atctgcgcgc gaggccaagc gatcttcttc ttgtccaaga taagcctgtc tagcttcaag
      121 tatgacgggc tgatactggg ccggcaggcg ctccattgcc cagtcggcag cgacatcctt
      181 aggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt
      241 tcgctcatcg ccagcccagt cgggcggcga gttccatagc gttaaggttt catttagcgc
      301 ctcaaataga tcctgttcag gaaccggatc aaagagttcc tccgccgctg gacctaccaa
      361 ggcaacgcta tgttctcttg cttttgtcag caagatagcc agatcaatgt cgatcgtggc
      421 tggctcgaag atacctgcaa gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg
      481 cttagctgga taacgccaag gaatgatgtc gtcgtgcaca acaatggtga cttctacagc
      541 gcggagaatc tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt tgatcaaagc
      601 tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc agcaaatcaa tatcactgtg
      661 tggcttcagg ccgccatcca ctgcggagcc gtacaaatgt acggccagca acgtcggttc
      721 gagatggcgc tcgatgacgc caactacctc tgatagttga gtcgatactt cggcgatcac
      781 cgcttccctc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
      841 catgagcgga tacatatttg aatgtattta gaaaaatagg ccaaataggc cgttgagatc
      901 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
      961 tttgtttgcc ggatcaagag ctaccacctc tttttccgaa ggtaactggc ttcagcagag
     1021 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     1081 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     1141 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     1201 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     1261 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     1321 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     1381 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     1441 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     1501 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     1561 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     1621 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     1681 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
     1741 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     1801 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     1861 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     1921 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     1981 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     2041 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     2101 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     2161 gaggatgctc acgatacggg ttactgatga tgaacaagag gacatccggt caaataaaac
     2221 gaaaggctca gtcgaaagac tgggcctttc gttttgctga ggatggaaag aagagaaacc
     2281 tgattattcg ttttgctgag gagacttagg gaccctacaa cacgcacggt gttacattag
     2341 gcaccccggg ctttacactt tatgcttccg gctcgtatgt tgtgtcgacc gacctgcagg
     2401 tgcagtaaaa aaaaaggaaa atgaaaaaat tattattcgc aattccttta gttgttcctt
     2461 tctattctca ctccgctgaa actgttgaaa gttgtttagc aaaaccccat acagaaaatt
     2521 catttactaa cgtctggaaa gacgacaaaa ctttagatcg ttacgctaac tatgagggct
     2581 gtctgtggaa tgctacaggc gttgtagttt gtactggtga cgaaactcag tgttacggta
     2641 catgggttcc tattgggctt gctatccctg aaaatgaggg tggtggctct gagggtggcg
     2701 gttctgaggg tggcggttct gagggtggcg gtactaaacc tcctgagtac ggtgatacac
     2761 ctattccggg ctatacttat atcaaccctc tcgacggcac ttatccgcct ggtactgagc
     2821 aaaaccccgc taatcctaat ccttctcttg aggagtctca gcctcttaat actttcatgt
     2881 ttcagaataa taggttccga aataggcagg gggcattaac tgtttatacg ggcactgtta
     2941 ctcaaggcac tgaccccgtt aaaacttatt accagtacac tcctgtatca tcaaaagcca
     3001 tgtatgacgc ttactggaac ggtaaattca gagactgcgc tttccattct ggctttaatg
     3061 aggatccatt cgtttgtgaa tatcaaggcc aatcgtctga cctgcctcaa cctcctgtca
     3121 atgctggcgg cggctctggt ggtggttctg gtggcggctc tgagggtggt ggctctgagg
     3181 gtggcggttc tgagggtggc ggctctgagg gaggcggttc cggtggtggc tctggttccg
     3241 gtgattttga ttatgaaaag atggcaaacg ctaataaggg ggctatgacc gaaaatgccg
     3301 atgaaaacgc gctacagtct gacgctaaag gcaaacttga ttctgtcgct actgattacg
     3361 gtgctgctat cgatggtttc attggtgacg tttccggcct tgctaatggt aatggtgcta
     3421 ctggtgattt tgctggctct aattcccaaa tggctcaagt cggtgacggt gataattcac
     3481 ctttaatgaa taatttccgt caatatttac cttccctccc tcaatcggtt gaatgtcgcc
     3541 cttttgtctt tggcgctggt aaaccttacg agttcagtat cgactgcgat aagatcaacc
     3601 tgttccgcgg tgtctttgcg tttcttttat atgttgccac ctttatgtat gtattttcta
     3661 cgtttgctaa catactgcgt aataaggagt cttaatgaaa tttggaaact ttttgcttac
     3721 ataccaacct ccccaatttt cccaaacaga ggtaatgaaa cgtttggtta aattaggtcg
     3781 catctctgag gagtgtggtt ttgataccgt atggttactg gagcatcatt tcacggagtt
     3841 tggtttgctt ggtaaccctt atgtcgctgc tgcatattta cttggcgcga ctaaaaaatt
     3901 gaatgtagga actgccgcta ttgttcttcc cacagcccat ccagtacgcc aacttgaaga
     3961 tgtgaattta ttggatcaaa tgtcaaaagg acgatttcgg tttggtattt gccgagggct
     4021 ttacaacaag gactttcgcg tattcggcac agatatgaat aacagtcgcg ccttagcgga
     4081 atgctggtac gggctgataa agaatggcat gacagaggga tatatggaag ctgataatga
     4141 acatatcaag ttccataagg taaaagtaaa ccccgcggcg tatagcagag gtggcgcacc
     4201 ggtttatgtg gtggctgaat cagcttcgac gactgagtgg gctgctcaat ttggcctacc
     4261 gatgatatta agttggatta taaatactaa cgaaaagaaa gcacaacttg agctttataa
     4321 tgaagtggct caagaatatg ggcacgatat tcataatatc gaccattgct tatcatatat
     4381 aacatctgta gatcatgact caattaaagc gaaagagatt tgccggaaat ttctggggca
     4441 ttggtatgat tcttatgtga atgctacgac tatttttgat gattcagacc aaacaagagg
     4501 ttatgatttc aataaagggc agtggcgtga ctttgtatta aaaggacata aagatactaa
     4561 tcgccgtatt gattacagtt acgaaatcaa tcccgtggga acgccgcagg aatgtattga
     4621 cataattcaa aaagacattg atgctacagg aatatcaaat atttgttgtg gatttgaagc
     4681 taatggaaca gtagacgaaa ttattgcttc catgaagctc ttccagtctg atgtcatgcc
     4741 atttcttaaa gaaaaacaac gttcgctatt atattatggc ggtggcggta gcggcggtgg
     4801 cggtagcggc ggtggcggta gcggcggtgg cggtagcaaa tttggattgt tcttccttaa
     4861 cttcatcaat tcaacaactg ttcaagaaca gagtatagtt cgcatgcagg aaataacgga
     4921 gtatgttgat aagttgaatt ttgaacagat tttagtgtat gaaaatcatt tttcagataa
     4981 tggtgttgtc ggcgctcctc tgactgtttc tggttttctg ctcggtttaa cagagaaaat
     5041 taaaattggt tcattaaatc acatcattac aactcatcat cctgtccgca tagcggagga
     5101 agcttgctta ttggatcagt taagtgaagg gagatttatt ttagggttta gtgattgcga
     5161 aaaaaaagat gaaatgcatt tttttaatcg cccggttgaa tatcaacagc aactatttga
     5221 agagtgttat gaaatcatta acgatgcttt aacaacaggc tattgtaatc cagataacga
     5281 tttttatagc ttccctaaaa tatctgtaaa tccccatgct tatacgccag gcggacctcg
     5341 gaaatatgta acagcaacca gtcatcatat tgttgagtgg gcggccaaaa aaggtattcc
     5401 tctcatcttt aagtgggatg attctaatga tgttagatat gaatatgctg aaagatataa
     5461 agccgttgcg gataaatatg acgttgacct atcagagata gaccatcagt taatgatatt
     5521 agttaactat aacgaagata gtaataaagc taaacaagaa acacgtgcat ttattagtga
     5581 ttatgttctt gaaatgcacc ctaatgaaaa tttcgaaaat aaacttgaag aaataattgc
     5641 agaaaacgct gtcggaaatt atacggagtg tataactgcg gctaagttgg caattgaaaa
     5701 gtgtggtgcg aaaagtgtat tgctgtcctt tgaaccaatg aatgatttga tgagccaaaa
     5761 aaatgtaatc aatattgttg atgataatat taagaagtac cacacggaat atacctaaac
     5821 ggagccaatg tacgcaaaaa accccgcttc agcggggttt tttcgccaaa aaaaaccccg
     5881 ccctgtcagg ggcggggttt ttttttaggg ccctactgac tgtaaaaaaa gcaccgactc
     5941 ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta tttctagctc
     6001 taaaacttcg ttttgctgag gagactccac acaacatacg agccggaagc ataaagtgta
     6061 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     6121 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     6181 gaggcggttt gcgtattggg cgcacttaat taacggcact gcgaaaaaac cccgccgaag
     6241 cggggttttt tgcgaaatat aatgaccctc ttgataaccc aagagggcat tttttaatgc
     6301 ccatggcgt
//
