LOCUS       sequence-269263-        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"
     promoter        complement(656..747)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      756..1344
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1245..1264
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1498..1515
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1530..1670
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1596..1615)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1756..1778
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1772..1963)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        join(2215..2220,2221..2238,2239..2245)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             3558..4637
                     /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             4701..5681
                     /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(5794..5869)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(5895..5901,5902..5919,5920..5925))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5940..5961
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(join(6173..6309,1..655))
                     /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"
ORIGIN
        1 gggccggcag gcgctccatt gcccagtcgg cagcgacatc cttcggcgcg attttgccgg
       61 ttactgcgct gtaccaaatg cgggacaacg taagcactac atttcgctca tctccagccc
      121 agtcgggcgg cgagttccat agcgttaagg tttcatttag cgcctcaaat agatcctgtt
      181 caggaaccgg atcaaagagt tcctccgccg ctggacctac caaggcaacg ctatgttctc
      241 ttgcttttgt cagcaagata gccagatcaa tgtcgatcgt ggctggctcg aagatacctg
      301 caagaatgtc attgcgctgc cattctccaa attgcagttc gcgcttagct ggataacgcc
      361 acggaatgat gtcgtcgtgc acaacaatgg tgacttctac agcgcggaga atctcgctct
      421 ctccagggga agccgaagtt tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat
      481 caagccttac ggtcacagta accagcaaat caatatcact gtgtggcttc aggccgccat
      541 ccactgcgga gccgtacaaa tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga
      601 cgccaactac ctctgatagt tgagtcgata cttcggcgat caccgcttcc ctcatactct
      661 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
      721 ttgaatgtat ttagaaaaat aggccaaata ggccgttgag atcctttttt tctgcgcgta
      781 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
      841 gagctaccac ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
      901 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
      961 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1021 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     1081 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     1141 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     1201 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     1261 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     1321 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     1381 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     1441 cgtattacag cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     1501 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     1561 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     1621 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     1681 cgccaacacc cgctgacgcg ccctgacggg cttgtcttct cccggcatcc gcttacagac
     1741 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     1801 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     1861 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     1921 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     1981 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     2041 gggttactga tgatgaacaa gaggacatcc ggtcaaataa aacgaaaggc tcagtcgaaa
     2101 gactgggcct ttcgttttgc tgaggatgga aagaagagaa acctgattat tcgttttgct
     2161 gaggagactt agggacccta caacacgcac ggtgttacat taggcacccc gggctttaca
     2221 ctttatgctt ccggctcgta tgttgtgtcg accgacctgc aggtgcagta aaaaaaaagg
     2281 aaaatgaaaa aattattatt cgcaattcct ttagttgttc ctttctattc tcactccgcc
     2341 gaaactgttg aaagttgttt agcaaaaccc catacagaaa attcatttac taacgtctgg
     2401 aaagacgaca aaactttaga tcgttacgct aactatgagg gctgtctgtg gaatgctaca
     2461 ggcgttgtag tttgtactgg tgacgaaact cagtgttacg gtacatgggt tcctattggg
     2521 cttgctatcc ctgaaaatga gggtggtggc tctgagggtg gcggttctga gggtggcggt
     2581 tctgagggtg gcggtactaa acctcctgag tacggtgata cacctattcc gggctatact
     2641 tatatcaacc ctctcgacgg cacttatccg cctggtactg agcaaaaccc cgctaatcct
     2701 aatccttctc ttgaggagtc tcagcctctt aatactttca tgtttcagaa taataggttc
     2761 cgaaataggc agggggcatt aactgtttat acgggcactg ttactcaagg cactgacccc
     2821 gttaaaactt attaccagta cactcctgta tcatcaaaag ccatgtatga cgcttactgg
     2881 aacggtaaat tcagagactg cgctttccat tctggcttta atgaggatcc attcgtttgt
     2941 gaatatcaag gccaatcgtc tgacctgcct caacctcctg tcaatgctgg cggcggctct
     3001 ggtggtggtt ctggtggcgg ctctgagggt ggtggctctg agggtggcgg ttctgagggt
     3061 ggcggctctg agggaggcgg ttccggtggt ggctctggtt ccggtgattt tgattatgaa
     3121 aagatggcaa acgctaataa gggggctatg accgaaaatg ccgatgaaaa cgcgctacag
     3181 tctgacgcta aaggcaaact tgattctgtc gctactgatt acggtgctgc tatcgatggt
     3241 ttcattggtg acgtttccgg ccttgctaat ggtaatggtg ctactggtga ttttgctggc
     3301 tctaattccc aaatggctca agtcggtgac ggtgataatt cacctttaat gaataatttc
     3361 cgtcaatatt taccttccct ccctcaatcg gttgaatgtc gcccttttgt ctttggcgct
     3421 ggtaaacctt acgagttcag tatcgactgc gataagatca acctgttccg cggtgtcttt
     3481 gcgtttcttt tatatgttgc cacctttatg tatgtatttt ctacgtttgc taacatactg
     3541 cgtaataagg agtcttaatg aaatttggaa actttttgct tacataccaa cctccccaat
     3601 tttcccaaac agaggtaatg aaacgtttgg ttaaattagg tcgcatctct gaggagtgtg
     3661 gttttgatac cgtatggtta ctggagcatc atttcacgga gtttggtttg cttggtaacc
     3721 cttatgtcgc tgctgcatat ttacttggcg cgactaaaaa attgaatgta ggaactgccg
     3781 ctattgttct tcccacagcc catccagtac gccaacttga agatgtgaat ttattggatc
     3841 aaatgtcaaa aggacgattt cggtttggta tttgccgagg gctttacaac aaggactttc
     3901 gcgtattcgg cacagatatg aataacagtc gcgccttagc ggaatgctgg tacgggctga
     3961 taaagaatgg catgacagag ggatatatgg aagctgataa tgaacatatc aagttccata
     4021 aggtaaaagt aaaccccgcg gcgtatagca gaggtggcgc accggtttat gtggtggctg
     4081 aatcagcttc gacgactgag tgggctgctc aatttggcct accgatgata ttaagttgga
     4141 ttataaatac taacgaaaag aaagcacaac ttgagcttta taatgaagtg gctcaagaat
     4201 atgggcacga tattcataat atcgaccatt gcttatcata tataacatct gtagatcatg
     4261 actcaattaa agcgaaagag atttgccgga aatttctggg gcattggtat gattcttatg
     4321 tgaatgctac gactattttt gatgattcag accaaacaag aggttatgat ttcaataaag
     4381 ggcagtggcg tgactttgta ttaaaaggac ataaagatac taatcgccgt attgattaca
     4441 gttacgaaat caatcccgtg ggaacgccgc aggaatgtat tgacataatt caaaaagaca
     4501 ttgatgctac aggaatatca aatatttgtt gtggatttga agctaatgga acagtagacg
     4561 aaattattgc ttccatgaag ctcttccagt ctgatgtcat gccatttctt aaagaaaaac
     4621 aacgttcgct attatattat ggcggtggcg gtagcggcgg tggcggtagc ggcggtggcg
     4681 gtagcggcgg tggcggtagc aaatttggat tgttcttcct taacttcatc aattcaacaa
     4741 ctgttcaaga acagagtata gttcgcatgc aggaaataac ggagtatgtt gataagttga
     4801 attttgaaca gattttagtg tatgaaaatc atttttcaga taatggtgtt gtcggcgctc
     4861 ctctgactgt ttctggtttt ctgctcggtt taacagagaa aattaaaatt ggttcattaa
     4921 atcacatcat tacaactcat catcctgtcc gcatagcgga ggaagcttgc ttattggatc
     4981 agttaagtga agggagattt attttagggt ttagtgattg cgaaaaaaaa gatgaaatgc
     5041 atttttttaa tcgcccggtt gaatatcaac agcaactatt tgaagagtgt tatgaaatca
     5101 ttaacgatgc tttaacaaca ggctattgta atccagataa cgatttttat agcttcccta
     5161 aaatatctgt aaatccccat gcttatacgc caggcggacc tcggaaatat gtaacagcaa
     5221 ccagtcatca tattgttgag tgggcggcca aaaaaggtat tcctctcatc tttaagtggg
     5281 atgattctaa tgatgttaga tatgaatatg ctgaaagata taaagccgtt gcggataaat
     5341 atgacgttga cctatcagag atagaccatc agttaatgat attagttaac tataacgaag
     5401 atagtaataa agctaaacaa gaaacacgtg catttattag tgattatgtt cttgaaatgc
     5461 accctaatga aaatttcgaa aataaacttg aagaaataat tgcagaaaac gctgtcggaa
     5521 attatacgga gtgtataact gcggctaagt tggcaattga aaagtgtggt gcgaaaagtg
     5581 tattgctgtc ctttgaacca atgaatgatt tgatgagcca aaaaaatgta atcaatattg
     5641 ttgatgataa tattaagaag taccacacgg aatataccta aacggagcca atgtacgcaa
     5701 aaaaccccgc ttcagcgggg ttttttcgcc aaaaaaaacc ccgccctgtc aggggcgggg
     5761 ttttttttta gggccctact gactgtaaaa aaagcaccga ctcggtgcca ctttttcaag
     5821 ttgataacgg actagcctta ttttaacttg ctatttctag ctctaaaact tcgttttgct
     5881 gaggagactc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5941 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     6001 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6061 gggcgcactt aattaacggc actgcgaaaa aaccccgccg aagcggggtt ttttgcgaaa
     6121 tataatgacc ctcttgataa cccaagaggg cattttttaa tgcccatggc gtttatttgc
     6181 cgactacctt ggtgatctcg cctttcacgt agtggacaaa ttcttccaac tgatctgcgc
     6241 gcgaggccaa gcgatcttct tcttgtccaa gataagcctg tctagcttca agtatgacgg
     6301 gctgatact
//
