LOCUS       sequence-131909-        7092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for SaCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7092)
  AUTHORS   Kleinstiver BP, Prew MS, Tsai SQ, Nguyen NT, Topkar VV, Zheng Z, 
            Joung JK
  TITLE     Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 
            by modifying PAM recognition.
  JOURNAL   Nat Biotechnol. 2015 Nov 2. doi: 10.1038/nbt.3404.
  PUBMED    26524662
REFERENCE   2  (bases 1 to 7092)
  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: "Nat 
            Biotechnol. 2015 Nov 2. doi: 10.1038/nbt.3404."
COMMENT     Sequence Label: MSP2266
FEATURES             Location/Qualifiers
     source          1..7092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             71..3232
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENN
                     EGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEE
                     EFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDG
                     EVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPF
                     GWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK
                     FQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARK
                     EIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLK
                     AINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQS
                     IKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKEN
                     AKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLV
                     KQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINR
                     FSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKE
                     RNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKE
                     IFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDK
                     DNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSK
                     KDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVK
                     NLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDL
                     LNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHP
                     QIIKKG"
     CDS             3239..3259
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3266..3331
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        3401..3419
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3531..3549)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3545..3592
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3813..4472)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4387..4406
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4473..4575)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4967..4984)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5101..5645)
                     /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(5181..5200)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5870..6952)
                     /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     6914..6933
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6953..7030)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(7076..7092,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcaaac ggaactacat cctggggctt gacattggga taaccagcgt
      121 tggctacgga attattgatt atgagacacg cgatgtgatt gacgccgggg ttaggctgtt
      181 caaagaggcc aacgttgaaa acaacgaggg aagacggagt aagcgcggag caagaagact
      241 caagcgcaga cggagacatc ggattcagag ggtgaaaaag ctgctcttcg attacaatct
      301 cctgaccgat catagtgagc tgagcggaat caacccctac gaggcgcgag tgaaagggct
      361 ttcccagaag ctgtccgaag aggagttctc cgccgcgttg ctgcacctgg ccaaacggag
      421 gggggttcac aatgtaaacg aagtggagga ggacacgggc aatgaactta gtacgaaaga
      481 acagatcagt aggaactcta aggctctcga agagaaatac gtcgctgagt tgcagcttga
      541 gagactgaaa aaagacggcg aagtacgcgg atctattaat aggttcaaga cttcagatta
      601 cgtaaaggaa gccaagcagc tcctgaaagt acagaaagcg taccatcagc tcgatcagag
      661 cttcatcgat acctacatag atttgctgga gacacggagg acatactacg agggcccagg
      721 ggaaggatct ccttttgggt ggaaggacat caaggaatgg tacgagatgc ttatgggaca
      781 ttgtacatat tttccggagg agctcaggag cgtcaagtac gcctacaatg ccgacctgta
      841 caatgccctc aatgacctca ataacctcgt gattaccagg gacgagaacg agaagctgga
      901 gtactatgaa aagttccaga ttatcgagaa tgtgtttaag cagaagaaga agccgacact
      961 taagcagatt gcaaaggaaa tcctcgtgaa tgaggaagat atcaagggat acagagtgac
     1021 aagtacaggc aagcccgagt tcacaaatct gaaggtgtac cacgatatta aggacataac
     1081 cgcacgaaag gagataatcg aaaacgctga gctcctcgat cagatcgcaa aaattcttac
     1141 catctaccag tctagtgagg acattcagga ggaactgact aatctgaaca gtgagctcac
     1201 ccaagaggaa attgagcaga tttcaaacct gaaaggctac accgggacgc acaatctgag
     1261 cctcaaagca atcaacctca ttctggatga actttggcac acaaatgaca accaaattgc
     1321 catattcaac cgcctgaaac tggtgccaaa aaaagtggat ctgtcacagc aaaaggaaat
     1381 ccctacaacc ttggttgacg attttattct gtcccccgtt gtcaagcgga gcttcatcca
     1441 gtcaatcaag gtgatcaatg ccatcattaa aaaatacgga ttgccaaacg atataattat
     1501 cgagcttgca cgagagaaga actcaaagga cgcccagaag atgattaacg aaatgcagaa
     1561 gcgcaaccgc cagacaaacg aacgcataga ggaaattata agaacaaccg gcaaagagaa
     1621 tgccaagtat ctgatcgaga aaatcaagct gcacgacatg caagaaggca agtgcctgta
     1681 ctctctggaa gctatcccac tcgaagatct gctgaataat ccattcaatt acgaggtgga
     1741 ccacatcatc cctagatccg taagctttga caattccttc aataacaaag ttctggttaa
     1801 acaggaggaa aattctaaaa aagggaaccg gaccccgttc cagtacctga gctccagtga
     1861 cagcaagatt agctacgaga cttttaagaa acatattctg aatctggcca aaggcaaagg
     1921 caggatcagc aagaccaaga aggagtacct cctcgaagaa cgcgacatta acagatttag
     1981 tgtgcagaaa gatttcatca accgaaacct tgtcgatact cggtacgcca cgagaggcct
     2041 gatgaatctc ctcaggagct acttccgcgt caataatctg gacgttaaag tcaagagcat
     2101 aaatggggga ttcaccagct ttctgaggag aaagtggaag tttaagaagg aacgaaacaa
     2161 aggatacaag caccatgctg aggatgcttt gatcatcgct aacgcggact ttatctttaa
     2221 ggaatggaaa aagctggata aggcaaagaa agtgatggaa aaccagatgt tcgaggagaa
     2281 gcaggcagag tcaatgcctg agatcgagac agagcaggaa tacaaggaaa ttttcatcac
     2341 ccctcatcag attaaacaca taaaggactt caaagactat aaatactctc atagggtgga
     2401 caaaaaaccc aatcgcgagc tcattaatga caccctgtac tcaacacgga aggatgataa
     2461 aggtaatacc ttgattgtga ataatcttaa tggattgtat gacaaagata acgacaagct
     2521 caagaagctg atcaacaagt ctccagagaa gctccttatg tatcaccacg acccacagac
     2581 ttatcagaaa ttgaaactga tcatggagca atacggggat gagaagaacc cactctacaa
     2641 atattatgag gaaacaggta attacctgac caagtactcc aagaaggata acggaccagt
     2701 gatcaaaaag ataaagtact atggcaacaa acttaatgcg catttggaca taactgacga
     2761 ttaccccaat tctcgaaaca aggttgtgaa gctctccctg aagccttata gatttgacgt
     2821 gtacctggat aatggggttt ataaattcgt caccgtgaaa aatctggacg tgatcaaaaa
     2881 ggagaactat tatgaagtaa actcaaagtg ctatgaggag gcgaagaagc tgaagaagat
     2941 ctccaatcag gccgagttca tcgcttcctt ctataataac gatctcatca agatcaatgg
     3001 agagctttat cgcgtcattg gtgtgaacaa tgacttgctg aacaggatcg aagtcaatat
     3061 gatagacatt acctaccggg agtatctcga aaacatgaat gataaacggc cgcctcgcat
     3121 catcaagaca atcgcatcta aaactcagtc aataaaaaag tactctaccg atatcctggg
     3181 gaatctctat gaagtgaagt caaagaagca cccacaaatc attaaaaaag gtggatcccc
     3241 caagaagaag aggaaagtct cgagcgacta caaagaccat gacggtgatt ataaagatca
     3301 tgacatcgat tacaaggatg acgatgacaa gtaaagcggc cgcataatgc ttaagtcgaa
     3361 cagaaagtaa tcgtattgta cacggccgca taatcgaaat taatacgact cactataggt
     3421 cgccctcgaa cttcacctgt tttagtactc tgtaatgaaa attacagaat ctactaaaac
     3481 aaggcaaaat gccgtgttta tctcgtcaac ttgttggcga gatttttttt ccgctgagca
     3541 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaacc
     3601 tcaggcattt gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca
     3661 gcaatagaca taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcgaattt
     3721 gctttcgaat ttctgccatt catccgctta ttatcactta ttcaggcgta gcaaccaggc
     3781 gtttaagggc accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag
     3841 tactgttgta attcattaag cattctgccg acatggaagc catcacagac ggcatgatga
     3901 acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatagtg
     3961 aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc
     4021 acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc
     4081 aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg
     4141 tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa
     4201 caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat acggaactcc
     4261 ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta
     4321 tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta
     4381 cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca
     4441 acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc
     4501 gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct
     4561 cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg cccagggctt cccggtatca
     4621 acagggacac caggatttat ttattctgcg aagtgatctt ccgtcacagg tatttattcg
     4681 gcgcaaagtg cgtcgggtga tgctgccaac ttactgattt agtgtatgat ggtgtttttg
     4741 aggtgctcca gtggcttctg tttctatcag ctgtccctcc tgttcagcta ctgacggggt
     4801 ggtgcgtaac ggcaaaagca ccgccggaca tcagcgctag cggagtgtat actggcttac
     4861 tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg
     4921 caccggtgcg tcagcagaat atgtgataca ggatatattc cgcttcctcg ctcactgact
     4981 cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt
     5041 tcctggaaga tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt
     5101 tttccatagg ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg
     5161 gcgaaacccg acaggactat aaagatacca ggcgtttccc ctggcggctc cctcgtgcgc
     5221 tctcctgttc ctgcctttcg gtttaccggt gtcattccgc tgttatggcc gcgtttgtct
     5281 cattccacgc ctgacactca gttccgggta ggcagttcgc tccaagctgg actgtatgca
     5341 cgaacccccc gttcagtccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5401 cccggaaaga catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt
     5461 tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg
     5521 ctcctccaag ccagttacct cggttcaaag agttggtagc tcagagaacc ttcgaaaaac
     5581 cgccctgcaa ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat
     5641 ctcaagaaga tcatcttatt aatcagataa aatatttcta gatttcagtg caatttatct
     5701 cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt
     5761 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     5821 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     5881 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5941 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     6001 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     6061 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     6121 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     6181 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     6241 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     6301 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     6361 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     6421 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     6481 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     6541 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     6601 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     6661 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     6721 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     6781 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     6841 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     6901 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     6961 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     7021 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaaattaata
     7081 cgactcacta ta
//
