LOCUS       sequence-447614-        7952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual pT7 entry plasmid for SpCas9(6AA) library; bacterial expression
            plasmid with type IIS RE cassettes around D1135/S1136, G1218/E1219, 
            R1335/T1337 (precursor to MMW94). Expresses a gRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7952)
  AUTHORS   Silverstein RA, Kim N, Kroell AS, Walton RT, Delano J, Butcher RM, 
            Pacesa M, Smith BK, Christie KA, Ha LL, Meis RJ, Clark AB, Spinner 
            AD, Lazzarotto CR, Li Y, Matsubara A, Urbina EO, Dahl GA, Correia 
            BE, Marks DS, Tsai SQ, Pinello L, De Ravin SS, Liu Q, Kleinstiver BP
  TITLE     Custom CRISPR-Cas9 PAM variants via scalable engineering and machine
            learning.
  JOURNAL   Nature. 2025 Apr 22. doi: 10.1038/s41586-025-09021-y.
  PUBMED    40262634
REFERENCE   2  (bases 1 to 7952)
  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: "Nature. 
            2025 Apr 22. doi: 10.1038/s41586-025-09021-y."
COMMENT     Sequence Label: pACYC-T7-SpCas9(BspMI/SapI/BsaI_cassettes)-T7-...
FEATURES             Location/Qualifiers
     source          1..7952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    2..26
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             70..1788
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             4111..4131
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4138..4203
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DNKDHDGDYKDHDIDYKDDDDK"
     promoter        4273..4291
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        4312..4387
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(4389..4407)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4403..4450
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4671..5330)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5245..5264
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5331..5433)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5825..5842)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5959..6504)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(6039..6058)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6729..7811)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7773..7792
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7812..7889)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(7935..7952,1)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggaattgtg agcggataac aattcccctg tagaaataat tttgtttaac tttaataagg
       61 agatatacca tggataaaaa gtattctatt ggtttagaca tcggcactaa ttccgttgga
      121 tgggctgtca taaccgatga atacaaagta ccttcaaaga aatttaaggt gttggggaac
      181 acagaccgtc attcgattaa aaagaatctt atcggtgccc tcctattcga tagtggcgaa
      241 acggcagagg cgactcgcct gaaacgaacc gctcggagaa ggtatacacg tcgcaagaac
      301 cgaatatgtt acttacaaga aatttttagc aatgagatgg ccaaagttga cgattctttc
      361 tttcaccgtt tggaagagtc cttccttgtc gaagaggaca agaaacatga acggcacccc
      421 atctttggaa acatagtaga tgaggtggca tatcatgaaa agtacccaac gatttatcac
      481 ctcagaaaaa agctagttga ctcaactgat aaagcggacc tgaggttaat ctacttggct
      541 cttgcccata tgataaagtt ccgtgggcac tttctcattg agggtgatct aaatccggac
      601 aactcggatg tcgacaaact gttcatccag ttagtacaaa cctataatca gttgtttgaa
      661 gagaacccta taaatgcaag tggcgtggat gcgaaggcta ttcttagcgc ccgcctctct
      721 aaatcccgac ggctagaaaa cctgatcgca caattacccg gagagaagaa aaatgggttg
      781 ttcggtaacc ttatagcgct ctcactaggc ctgacaccaa attttaagtc gaacttcgac
      841 ttagctgaag atgccaaatt gcagcttagt aaggacacgt acgatgacga tctcgacaat
      901 ctactggcac aaattggaga tcagtatgcg gacttatttt tggctgccaa aaaccttagc
      961 gatgcaatcc tcctatctga catactgaga gttaatactg agattaccaa ggcgccgtta
     1021 tccgcttcaa tgatcaaaag gtacgatgaa catcaccaag acttgacact tctcaaggcc
     1081 ctagtccgtc agcaactgcc tgagaaatat aaggaaatat tctttgatca gtcgaaaaac
     1141 gggtacgcag ggtatattga cggcggagcg agtcaagagg aattctacaa gtttatcaaa
     1201 cccatattag agaagatgga tgggacggaa gagttgcttg taaaactcaa tcgcgaagat
     1261 ctactgcgaa agcagcggac tttcgacaac ggtagcattc cacatcaaat ccacttaggc
     1321 gaattgcatg ctatacttag aaggcaggag gatttttatc cgttcctcaa agacaatcgt
     1381 gaaaagattg agaaaatcct aacctttcgc ataccttact atgtgggacc cctggcccga
     1441 gggaactctc ggttcgcatg gatgacaaga aagtccgaag aaacgattac tccctggaat
     1501 tttgaggaag ttgtcgataa aggtgcgtca gctcaatcgt tcatcgagag gatgaccaac
     1561 tttgacaaga atttaccgaa cgaaaaagta ttgcctaagc acagtttact ttacgagtat
     1621 ttcacagtgt acaatgaact cacgaaagtt aagtatgtca ctgagggcat gcgtaaaccc
     1681 gcctttctaa gcggagaaca gaagaaagca atagtagatc tgttattcaa gaccaaccgc
     1741 aaagtgacag ttaagcaatt gaaagaggac tactttaaga aaattgaatg cttcgattct
     1801 gtcgagatct ccggggtaga agatcgattt aatgcgtcac ttggtacgta tcatgacctc
     1861 ctaaagataa ttaaagataa ggacttcctg gataacgaag agaatgaaga tatcttagaa
     1921 gatatagtgt tgactcttac cctctttgaa gatcgggaaa tgattgagga aagactaaaa
     1981 acatacgctc acctgttcga cgataaggtt atgaaacagt taaagaggcg tcgctatacg
     2041 ggctggggac gattgtcgcg gaaacttatc aacgggataa gagacaagca aagtggtaaa
     2101 actattctcg attttctaaa gagcgacggc ttcgccaata ggaactttat gcagctgatc
     2161 catgatgact ctttaacctt caaagaggat atacaaaagg cacaggtttc cggacaaggg
     2221 gactcattgc acgaacatat tgcgaatctt gctggttcgc cagccatcaa aaagggcata
     2281 ctccagacag tcaaagtagt ggatgagcta gttaaggtca tgggacgtca caaaccggaa
     2341 aacattgtaa tcgagatggc acgcgaaaat caaacgactc agaaggggca aaaaaacagt
     2401 cgagagcgga tgaagagaat agaagagggt attaaagaac tgggcagcca gatcttaaag
     2461 gagcatcctg tggaaaatac ccaattgcag aacgagaaac tttacctcta ttacctacaa
     2521 aatggaaggg acatgtatgt tgatcaggaa ctggacataa accgtttatc tgattacgac
     2581 gtcgatcaca ttgtacccca atcctttttg aaggacgatt caatcgacaa taaagtgctt
     2641 acacgctcgg ataagaaccg agggaaaagt gacaatgttc caagcgagga agtcgtaaag
     2701 aaaatgaaga actattggcg gcagctccta aatgcgaaac tgataacgca aagaaagttc
     2761 gataacttaa ctaaagctga gaggggtggc ttgtctgaac ttgacaaggc cggatttatt
     2821 aaacgtcagc tcgtggaaac ccgccaaatc acaaagcatg ttgcacagat actagattcc
     2881 cgaatgaata cgaaatacga cgagaacgat aagctgattc gggaagtcaa agtaatcact
     2941 ttaaagtcaa aattggtgtc ggacttcaga aaggattttc aattctataa agttagggag
     3001 ataaataact accaccatgc gcacgacgct tatcttaatg ccgtcgtagg gaccgcactc
     3061 attaagaaat acccgaagct agaaagtgag tttgtgtatg gtgattacaa agtttatgac
     3121 gtccgtaaga tgatcgcgaa aagcgaacag gagataggca aggctacagc caaatacttc
     3181 ttttattcta acattatgaa tttctttaag acggaaatca ctctggcaaa cggagagata
     3241 cgcaaacgac ctttaattga aaccaatggg gagacaggtg aaatcgtatg ggataagggc
     3301 cgggacttcg cgacggtgag aaaagttttg tccatgcccc aagtcaacat agtaaagaaa
     3361 actgaggtgc agaccggagg gttttcaaag gaatcgattc ttccaaaaag gaatagtgat
     3421 aagctcatcg ctcgtaaaaa ggactgggtc ggcaggttaa gtgaaagctt taagtgaacc
     3481 tgcgtcggta gtggcaaaag ttgagaaggg aaaatccaag aaactgaagt cagtcaaaga
     3541 attattgggg ataacgatta tggagcgctc gtcttttgaa aagaacccca tcgacttcct
     3601 tgaggcgaaa ggttacaagg aagtaaaaaa ggatctcata attaaactac caaagtatag
     3661 tctgtttgag ttagaaaatg gcgaagagct aagtgaggta cctaagtgag ctcttccacc
     3721 gtctaaatac gtgaatttcc tgtatttagc gtcccattac gagaagttga aaggttcacc
     3781 tgaagataac gaacagaagc aactttttgt tgagcagcac aaacattatc tcgacgaaat
     3841 catagagcaa atttcggaat tcagtaagag agtcatccta gctgatgcca atctggacaa
     3901 agtattaagc gcatacaaca agcacaggga taaacccata cgtgagcagg cggaaaatat
     3961 tatccatttg tttactctta ccaacctcgg cgctccagcg gagacctaag tgaactagtt
     4021 aagtgaggtc tcgcgacact gattcaccaa tccatcacgg gattatatga aactcggata
     4081 gatttgtcac agcttggggg tgacggatcc cccaagaaga agaggaaagt ctcgagcgac
     4141 aacaaagacc atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac
     4201 aagtgaagcg gccgcataat gcttaagtcg aacagaaagt aatcgtattg tacacggccg
     4261 cataatcgaa attaatacga ctcactatag ggggcacggg cagcttgccg ggttttagag
     4321 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     4381 tcggtgctcc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     4441 gggttttttg ctgaaacctc aggcatttga gaagcacacg gtcacactgc ttccggtagt
     4501 caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc
     4561 gacgaccggg tcgaatttgc tttcgaattt ctgccattca tccgcttatt atcacttatt
     4621 caggcgtagc aaccaggcgt ttaagggcac caataactgc cttaaaaaaa ttacgccccg
     4681 ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac atggaagcca
     4741 tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta
     4801 taatatttgc ccatagtgaa aacgggggcg aagaagttgt ccatattggc cacgtttaaa
     4861 tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt ctcaataaac
     4921 cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga atatatgtgt
     4981 agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt ttcagtttgc
     5041 tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc accgtctttc
     5101 attgccatac ggaactccgg atgagcattc atcaggcggg caagaatgtg aataaaggcc
     5161 ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat atccagctga
     5221 acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg ttctttacga
     5281 tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat tttagcttcc
     5341 ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct tatttcatta
     5401 tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca aaagttggcc
     5461 cagggcttcc cggtatcaac agggacacca ggatttattt attctgcgaa gtgatcttcc
     5521 gtcacaggta tttattcggc gcaaagtgcg tcgggtgatg ctgccaactt actgatttag
     5581 tgtatgatgg tgtttttgag gtgctccagt ggcttctgtt tctatcagct gtccctcctg
     5641 ttcagctact gacggggtgg tgcgtaacgg caaaagcacc gccggacatc agcgctagcg
     5701 gagtgtatac tggcttacta tgttggcact gatgagggtg tcagtgaagt gcttcatgtg
     5761 gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat gtgatacagg atatattccg
     5821 cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt
     5881 acgaacgggg cggagatttc ctggaagatg ccaggaagat acttaacagg gaagtgagag
     5941 ggccgcggca aagccgtttt tccataggct ccgcccccct gacaagcatc acgaaatctg
     6001 acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     6061 tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg tcattccgct
     6121 gttatggccg cgtttgtctc attccacgcc tgacactcag ttccgggtag gcagttcgct
     6181 ccaagctgga ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc ttatccggta
     6241 actatcgtct tgagtccaac ccggaaagac atgcaaaagc accactggca gcagccactg
     6301 gtaattgatt tagaggagtt agtcttgaag tcatgcgccg gttaaggcta aactgaaagg
     6361 acaagttttg gtgactgcgc tcctccaagc cagttacctc ggttcaaaga gttggtagct
     6421 cagagaacct tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga gcaagagatt
     6481 acgcgcagac caaaacgatc tcaagaagat catcttatta atcagataaa atatttctag
     6541 atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag
     6601 ttgtaattct catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     6661 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     6721 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6781 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     6841 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     6901 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     6961 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca
     7021 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     7081 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     7141 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     7201 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     7261 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     7321 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     7381 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     7441 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     7501 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     7561 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     7621 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     7681 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     7741 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     7801 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     7861 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     7921 ctgcattagg aaattaatac gactcactat ag
//
