LOCUS       pET-28b-Cas9-His        9375 bp DNA     circular SYN 13-MAY-2021
DEFINITION  For in vitro expression  and purification of Cas9 protein.
ACCESSION   .
VERSION     .
KEYWORDS    pET-28b-Cas9-His
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9375)
  AUTHORS   Gagnon JA, Valen E, Thyme SB, Huang P, Ahkmetova L, Pauli A, 
            Montague TG, Zimmerman S, Richter C, Schier AF
  TITLE     Efficient mutagenesis by Cas9 protein-mediated oligonucleotide 
            insertion and large-scale assessment of single-guide RNAs.
  JOURNAL   PLoS One. 2014 May 29;9(5):e98186. doi: 
            10.1371/journal.pone.0098186. eCollection 2014.
  PUBMED    24873830
REFERENCE   2  (bases 1 to 9375)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9375)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2014-05-29"; pages: "
            10.1371/journal.pone.0098186. eCollection 2014"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    complement(5..144)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     230..252
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(249..437)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(1212..1233)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(1249..2328)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(2329..2406)
                     /label=lacI promoter
     primer_bind     2612..2631
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
     promoter        2715..2733
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2734..2758
                     /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             2773..2795
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2802..6905
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             6918..6938
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6954..6971
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      7038..7085
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      7122..7577
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(7672..8484)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      8606..9194
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9348..9365
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
       61 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
      121 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
      181 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
      241 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
      301 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
      361 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
      421 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
      481 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
      541 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
      601 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
      661 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
      721 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
      781 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
      841 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
      901 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
      961 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     1021 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     1081 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     1141 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     1201 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     1261 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1321 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     1381 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     1441 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     1501 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     1561 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     1621 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     1681 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     1741 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     1801 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     1861 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     1921 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     1981 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     2041 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     2101 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     2161 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     2221 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     2281 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     2341 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     2401 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     2461 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     2521 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     2581 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     2641 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     2701 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     2761 tctagaaata attttgttta actttaagaa ggagatatac catggacaag aagtactcca
     2821 ttgggctcga tatcggcaca aacagcgtcg gctgggccgt cattacggac gagtacaagg
     2881 tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata aagaagaacc
     2941 tcattggcgc cctcctgttc gactccgggg agacggccga agccacgcgg ctcaaaagaa
     3001 cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag gagatcttta
     3061 gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag tcctttttgg
     3121 tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg gacgaggtgg
     3181 cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta gacagtactg
     3241 ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa tttcggggac
     3301 acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa ctctttatcc
     3361 aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca tccggagttg
     3421 acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa aacctcatcg
     3481 cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc ctgtcactcg
     3541 ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag cttcaactga
     3601 gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc gaccagtacg
     3661 cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt gatattctgc
     3721 gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag cgctatgatg
     3781 agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg cctgagaagt
     3841 acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt gacggcggag
     3901 caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg gacggcaccg
     3961 aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc actttcgaca
     4021 atggaagcat cccccaccag attcacctgg gcgaactgca cgctatcctc aggcggcaag
     4081 aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc ctcacatttc
     4141 ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg tggatgactc
     4201 gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat aagggggcct
     4261 ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct aacgaaaagg
     4321 tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag ctcaccaagg
     4381 tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag cagaagaaag
     4441 ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag ctcaaagaag
     4501 actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg gaggatcgct
     4561 tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac aaggacttcc
     4621 tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt acgttgtttg
     4681 aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc gacgacaaag
     4741 tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca agaaaactga
     4801 tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt aagtccgatg
     4861 gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc tttaaggagg
     4921 acatccagaa agcacaagtt tctggccagg gggacagtct tcacgagcac atcgctaatc
     4981 ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc gtggatgaac
     5041 tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg gcccgagaga
     5101 accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg attgaagagg
     5161 gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac acccagcttc
     5221 agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac gtggatcagg
     5281 aactggacat caatcggctc tccgactacg acgtggatca tatcgtgccc cagtcttttc
     5341 tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat agagggaaga
     5401 gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg cggcagctgc
     5461 tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct gaacgaggtg
     5521 gcctgtctga gttggataaa gccggcttca tcaaaaggca gcttgttgag acacgccaga
     5581 tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac gatgaaaatg
     5641 acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc tcagatttca
     5701 gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat gcgcatgatg
     5761 cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag cttgaatctg
     5821 aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca aagtctgagc
     5881 aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg aattttttca
     5941 agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc gaaacaaacg
     6001 gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc cggaaggtcc
     6061 tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga ggcttctcca
     6121 aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa aaagattggg
     6181 accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta ctggttgtgg
     6241 ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca
     6301 caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag gcgaaaggat
     6361 ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc tttgagcttg
     6421 aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt aacgagctgg
     6481 cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa aagctcaaag
     6541 ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa cactaccttg
     6601 atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc gacgctaacc
     6661 tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg gagcaggcag
     6721 aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc ttcaagtact
     6781 tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg gacgccacac
     6841 tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct cagctcggtg
     6901 gagacagcag ggctgacccc aagaagaaga ggaaggtggc ggccgcactc gagcaccacc
     6961 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     7021 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     7081 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     7141 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     7201 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     7261 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     7321 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     7381 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     7441 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     7501 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     7561 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     7621 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     7681 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     7741 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     7801 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     7861 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     7921 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     7981 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     8041 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     8101 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     8161 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     8221 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     8281 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     8341 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     8401 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     8461 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     8521 ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     8581 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     8641 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     8701 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     8761 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     8821 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     8881 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     8941 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     9001 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     9061 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     9121 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     9181 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     9241 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     9301 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     9361 gcgaggaagc ggaag
//
