LOCUS       40924_360        7157 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial expression plasmid for S. aureus Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7157)
  AUTHORS   Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, 
            Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK
  TITLE     Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
  JOURNAL   Nature. 2015 Jun 22. doi: 10.1038/nature14592.
  PUBMED    26098369
REFERENCE   2  (bases 1 to 7157)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7157)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2015 Jun 22. doi: 10.1038/nature14592."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /label=lac operator
                     /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)."
     RBS             42..64
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             77..3232
                     /codon_start=1
                     /label=SaCas9
                     /note="Cas9 endonuclease from the Staphylococcus aureus
                     Type II CRISPR/Cas system"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     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
                     /label=3xFLAG
                     /note="three tandem FLAG(R) epitope tags, followed by an 
                     enterokinase cleavage site"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        3401..3419
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3610..3657
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3881..4537)
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(4538..4640)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5032..5049)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5166..5710)
                     /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."
     protein_bind    complement(5901..5922)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5938..7017)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7018..7095)
                     /label=lacI promoter
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 cactataggg
     3421 agacccatgc catagcgttg ttcggaacag attcaccaac acctagtggt ctccgtttta
     3481 gtactctgta attttaggta tgaggtagac gaaaattgta cttataccta aaattacaga
     3541 atctactaaa acaaggcaaa atgccgtgtt tatctcgtca acttgttggc gagatccgct
     3601 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     3661 aaacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta
     3721 aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg
     3781 aatttgcttt cgaatttctg ccattcatcc gcttattatc acttattcag gcgtagcaac
     3841 caggcgttta agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcat
     3901 cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat
     3961 gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca
     4021 tagtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga
     4081 aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat
     4141 aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga
     4201 aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg
     4261 tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgga
     4321 actccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt
     4381 gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat
     4441 aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata
     4501 tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa
     4561 atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg
     4621 aacctcttac gtgccgatca acgtctcatt ttcgccaaaa gttggcccag ggcttcccgg
     4681 tatcaacagg gacaccagga tttatttatt ctgcgaagtg atcttccgtc acaggtattt
     4741 attcggcgca aagtgcgtcg ggtgatgctg ccaacttact gatttagtgt atgatggtgt
     4801 ttttgaggtg ctccagtggc ttctgtttct atcagctgtc cctcctgttc agctactgac
     4861 ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc gctagcggag tgtatactgg
     4921 cttactatgt tggcactgat gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa
     4981 ggctgcaccg gtgcgtcagc agaatatgtg atacaggata tattccgctt cctcgctcac
     5041 tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg
     5101 agatttcctg gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag
     5161 ccgtttttcc ataggctccg cccccctgac aagcatcacg aaatctgacg ctcaaatcag
     5221 tggtggcgaa acccgacagg actataaaga taccaggcgt ttcccctggc ggctccctcg
     5281 tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat tccgctgtta tggccgcgtt
     5341 tgtctcattc cacgcctgac actcagttcc gggtaggcag ttcgctccaa gctggactgt
     5401 atgcacgaac cccccgttca gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5461 tccaacccgg aaagacatgc aaaagcacca ctggcagcag ccactggtaa ttgatttaga
     5521 ggagttagtc ttgaagtcat gcgccggtta aggctaaact gaaaggacaa gttttggtga
     5581 ctgcgctcct ccaagccagt tacctcggtt caaagagttg gtagctcaga gaaccttcga
     5641 aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa
     5701 acgatctcaa gaagatcatc ttattaatca gataaaatat ttctagattt cagtgcaatt
     5761 tatctcttca aatgtagcac ctgaagtcag ccccatacga tataagttgt aattctcatg
     5821 ttagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     5881 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     5941 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     6001 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     6061 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     6121 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     6181 gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga
     6241 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     6301 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     6361 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     6421 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     6481 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     6541 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     6601 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     6661 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     6721 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     6781 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     6841 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     6901 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     6961 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     7021 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     7081 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaaat
     7141 taatacgact cactata
//
