pCasSA

LOCUS       pCasSA               10240 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  CRISPR-based E. coli/Staphylococcus aureus temperature-sensitive 
            plasmid for genome editing in Staphylococcus aureus.
ACCESSION   .
VERSION     .
KEYWORDS    pCasSA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10240)
  AUTHORS   Chen W, Zhang Y, Yeo WS, Bae T, Ji Q
  TITLE     Rapid and Efficient Genome Editing in Staphylococcus aureus by Using
            an Engineered CRISPR/Cas9 System.
  JOURNAL   J Am Chem Soc. 2017 Mar 15;139(10):3790-3795. doi: 
            10.1021/jacs.6b13317. Epub 2017 Mar 2.
  PUBMED    28218837
REFERENCE   2  (bases 1 to 10240)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..10240
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      79..667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     568..587
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        805..880
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1121..5227
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"                    
     primer_bind     5324..5342
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5380..5402)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5502..5521
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5715..5737
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5729..5746
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5730..5746
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9125..9141
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9125..9141
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             9312..10106
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"                     
     primer_bind     complement(9366..9385)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     9976..9995
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      10137..10231
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 agtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
       61 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
      121 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
      181 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      241 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      301 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      361 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      421 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      481 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      541 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      601 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      661 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      721 tcacatgttc tttcctgcgt tgtcgacaga gtttgcaaaa tatacagggg attatatata
      781 atggaaacga gaccattggt ctcagtttta gagctagaaa tagcaagtta aaataaggct
      841 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttgag atctgtccat
      901 acccatggtc tagaatgctc gagtcagaaa aatatacctg tatctttttt caaaagcaaa
      961 catgctttgg gtaaacatgt aggtattaac gtcaatgcga caatagtagc attgattaaa
     1021 tgagaattag taagtgtttt acttactaaa ttttatttaa cctaaaaatg aaccacctgg
     1081 atgtgtggga ttaaaaagtg aagagaggag gacatatcac atggataaga aatactcaat
     1141 aggcttagat atcggcacaa atagcgtcgg atgggcggtg atcactgatg aatataaggt
     1201 tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc cacagtatca aaaaaaatct
     1261 tataggggct cttttatttg acagtggaga gacagcggaa gcgactcgtc tcaaacggac
     1321 agctcgtaga aggtatacac gtcggaagaa tcgtatttgt tatctacagg agattttttc
     1381 aaatgagatg gcgaaagtag atgatagttt ctttcatcga cttgaagagt cttttttggt
     1441 ggaagaagac aagaagcatg aacgtcatcc tatttttgga aatatagtag atgaagttgc
     1501 ttatcatgag aaatatccaa ctatctatca tctgcgaaaa aaattggtag attctactga
     1561 taaagcggat ttgcgcttaa tctatttggc cttagcgcat atgattaagt ttcgtggtca
     1621 ttttttgatt gagggagatt taaatcctga taatagtgat gtggacaaac tatttatcca
     1681 gttggtacaa acctacaatc aattatttga agaaaaccct attaacgcaa gtggagtaga
     1741 tgctaaagcg attctttctg cacgattgag taaatcaaga cgattagaaa atctcattgc
     1801 tcagctcccc ggtgagaaga aaaatggctt atttgggaat ctcattgctt tgtcattggg
     1861 tttgacccct aattttaaat caaattttga tttggcagaa gatgctaaat tacagctttc
     1921 aaaagatact tacgatgatg atttagataa tttattggcg caaattggag atcaatatgc
     1981 tgatttgttt ttggcagcta agaatttatc agatgctatt ttactttcag atatcctaag
     2041 agtaaatact gaaataacta aggctcccct atcagcttca atgattaaac gctacgatga
     2101 acatcatcaa gacttgactc ttttaaaagc tttagttcga caacaacttc cagaaaagta
     2161 taaagaaatc ttttttgatc aatcaaaaaa cggatatgca ggttatattg atgggggagc
     2221 tagccaagaa gaattttata aatttatcaa accaatttta gaaaaaatgg atggtactga
     2281 ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc aagcaacgga cctttgacaa
     2341 cggctctatt ccccatcaaa ttcacttggg tgagctgcat gctattttga gaagacaaga
     2401 agacttttat ccatttttaa aagacaatcg tgagaagatt gaaaaaatct tgacttttcg
     2461 aattccttat tatgttggtc cattggcgcg tggcaatagt cgttttgcat ggatgactcg
     2521 gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa gttgtcgata aaggtgcttc
     2581 agctcaatca tttattgaac gcatgacaaa ctttgataaa aatcttccaa atgaaaaagt
     2641 actaccaaaa catagtttgc tttatgagta ttttacggtt tataacgaat tgacaaaggt
     2701 caaatatgtt actgaaggaa tgcgaaaacc agcatttctt tcaggtgaac agaagaaagc
     2761 cattgttgat ttactcttca aaacaaatcg aaaagtaacc gttaagcaat taaaagaaga
     2821 ttatttcaaa aaaatagaat gttttgatag tgttgaaatt tcaggagttg aagatagatt
     2881 taatgcttca ttaggtacct accatgattt gctaaaaatt attaaagata aagatttttt
     2941 ggataatgaa gaaaatgaag atatcttaga ggatattgtt ttaacattga ccttatttga
     3001 agatagggag atgattgagg aaagacttaa aacatatgct cacctctttg atgataaggt
     3061 gatgaaacag cttaaacgtc gccgttatac tggttgggga cgtttgtctc gaaaattgat
     3121 taatggtatt agggataagc aatctggcaa aacaatatta gattttttga aatcagatgg
     3181 ttttgccaat cgcaatttta tgcagctgat ccatgatgat agtttgacat ttaaagaaga
     3241 cattcaaaaa gcacaagtgt ctggacaagg cgatagttta catgaacata ttgcaaattt
     3301 agctggtagc cctgctatta aaaaaggtat tttacagact gtaaaagttg ttgatgaatt
     3361 ggtcaaagta atggggcggc ataagccaga aaatatcgtt attgaaatgg cacgtgaaaa
     3421 tcagacaact caaaagggcc agaaaaattc gcgagagcgt atgaaacgaa tcgaagaagg
     3481 tatcaaagaa ttaggaagtc agattcttaa agagcatcct gttgaaaata ctcaattgca
     3541 aaatgaaaag ctctatctct attatctcca aaatggaaga gacatgtatg tggaccaaga
     3601 attagatatt aatcgtttaa gtgattatga tgtcgatcac attgttccac aaagtttcct
     3661 taaagacgat tcaatagaca ataaggtctt aacgcgttct gataaaaatc gtggtaaatc
     3721 ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa aactattgga gacaacttct
     3781 aaacgccaag ttaatcactc aacgtaagtt tgataattta acgaaagctg aacgtggagg
     3841 tttgagtgaa cttgataaag ctggttttat caaacgccaa ttggttgaaa ctcgccaaat
     3901 cactaagcat gtggcacaaa ttttggatag tcgcatgaat actaaatacg atgaaaatga
     3961 taaacttatt cgagaggtta aagtgattac cttaaaatct aaattagttt ctgacttccg
     4021 aaaagatttc caattctata aagtacgtga gattaacaat taccatcatg cccatgatgc
     4081 gtatctaaat gccgtcgttg gaactgcttt gattaagaaa tatccaaaac ttgaatcgga
     4141 gtttgtctat ggtgattata aagtttatga tgttcgtaaa atgattgcta agtctgagca
     4201 agaaataggc aaagcaaccg caaaatattt cttttactct aatatcatga acttcttcaa
     4261 aacagaaatt acacttgcaa atggagagat tcgcaaacgc cctctaatcg aaactaatgg
     4321 ggaaactgga gaaattgtct gggataaagg gcgagatttt gccacagtgc gcaaagtatt
     4381 gtccatgccc caagtcaata ttgtcaagaa aacagaagta cagacaggcg gattctccaa
     4441 ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt gctcgtaaaa aagactggga
     4501 tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct tattcagtcc tagtggttgc
     4561 taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt aaagagttac tagggatcac
     4621 aattatggaa agaagttcct ttgaaaaaaa tccgattgac tttttagaag ctaaaggata
     4681 taaggaagtt aaaaaagact taatcattaa actacctaaa tatagtcttt ttgagttaga
     4741 aaacggtcgt aaacggatgc tggctagtgc cggagaatta caaaaaggaa atgagctggc
     4801 tctgccaagc aaatatgtga attttttata tttagctagt cattatgaaa agttgaaggg
     4861 tagtccagaa gataacgaac aaaaacaatt gtttgtggag cagcataagc attatttaga
     4921 tgagattatt gagcaaatca gtgaattttc taagcgtgtt attttagcag atgccaattt
     4981 agataaagtt cttagtgcat ataacaaaca tagagacaaa ccaatacgtg aacaagcaga
     5041 aaatattatt catttattta cgttgacgaa tcttggagct cccgctgctt ttaaatattt
     5101 tgatacaaca attgatcgta aacgatatac gtctacaaaa gaagttttag atgccactct
     5161 tatccatcaa tccatcactg gtctttatga aacacgcatt gatttgagtc agctaggagg
     5221 tgactgatgg ctggttggcg tactgttgtg gtaaataccc atgggtatgg acagttttcc
     5281 cgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     5341 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     5401 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     5461 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     5521 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     5581 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     5641 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     5701 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgcca agcttgcatg
     5761 cctgcagaac ggattgttga tgattacgaa aatattaaga gcacagacta ttacacagaa
     5821 aatcaagaat taaaaaaacg tagagagagt ttgaaagaag tagtgaatac atggaaagag
     5881 gggtatcacg aaaaaagtaa agaggttaat aaattaaagc gagagaatga tagtttgaat
     5941 gagcagttga atgtatcaga gaaatttcaa gatagtacag tgactttata tcgtgctgcg
     6001 agggcgaatt tccctgggtt tgagaaaggg tttaataggc ttaaagagaa attctttaat
     6061 gattccaaat tcgagcgtgt gggacagttt atggatgttg tacaggataa tgtccagaag
     6121 gtcgatagaa agcgtgagaa acagcgtaca gacgatttag agatgtagag gtacttttat
     6181 gccgagaaaa ctttttgcgt gtgacagtcc ttaaaatata cttagagcgt aagcgaaagt
     6241 agtagcgaca gctattaact ttcggttgca aagctctagg atttttaatg gacgcagcgc
     6301 atcacacgca aaaaggaaat tggaataaat gcgaaatttg agatgttaat taaagacctt
     6361 tttgaggtct ttttttctta gatttttggg gttatttagg ggagaaaaca taggggggta
     6421 ctacgacctc ccccctaggt gtccattgtc cattgtccaa acaaataaat aaatattggg
     6481 tttttaatgt taaaaggttg ttttttatgt taaagtgaaa aaaacagatg ttgggaggta
     6541 cagtgatggt tgtagataga aaagaagaga aaaaagttgc tgttacttta agacttacaa
     6601 cagaagaaaa tgagatatta aatagaatca aagaaaaata taatattagc aaatcagatg
     6661 caaccggtat tctaataaaa aaatatgcaa aggaggaata cggtgcattt taaacaaaaa
     6721 aagatagaca gcactggcat gctgcctatc tatgactaaa ttttgttaaa tgtattagca
     6781 ccgttattat atcatgagcg aaaatgtaat aaaagaaact gaaaacaaga aaaattcaag
     6841 aggacgtaat tggacatttg ttttatatcc agaatcagca aaagccgagt ggttagagta
     6901 tttaaaagag ttacacattc aatttgtagt gtctccatta catgataggg atactgatac
     6961 agaagatagg atgaaaaaag agcattatca tattctagtg atgtatgagg gtaataaatc
     7021 ttatgaacag ataaaaataa ttacagaaga attgaatgcg actattccgc agattgcagg
     7081 aagtgtgaaa ggtcttgtga gatatatgct tcacatggac gatcctaata aatttaaata
     7141 tcaaaaagaa gatatgatag tttatggcgg tgtagatgtt gatgaattat taaagaaaac
     7201 aacaacagat agatataaat taattaaaga aatgattgag tttattgatg aacaaggaat
     7261 cgtagaattt aagagtttaa tggattatgc aatgaagttt aaatttgatg attggttccc
     7321 gcttttatgt gataactcgg cgtatgttat tcaagaatat ataaaatcaa atcggtataa
     7381 atctgaccga tagattttga atttaagagt gtcacaagac actctttttt cgcaccagcg
     7441 aaaactggtt taagccgact gcgcaaaaga cataatcgat tcacaaaaaa taggcacacg
     7501 aaaaacaagt taagggatgc agtttatgca tcccttaact tacttattaa ataatttata
     7561 gctattgaaa agagataaga attgttcaaa gctaatattg tttaaatcgt caattcctgc
     7621 atgttttaag gaattgttaa attgattttt tgtaaatatt ttcttgtatt ctttgttatc
     7681 ttggttaccc gtcttcttaa tatgcgtaat tgcatctact ctaaatccat caatgccttt
     7741 atcaaaccac cagttcatca tttcaaatac agcatctcta acttccggat taccccaatt
     7801 caaatcaggt tgttttttac tgaataaatg gaaataatat tgctcagtat tagcatcata
     7861 ttcccgcgcg ccaattgagc tcccctttct atgtatgttt tttactagtc atttaaaacg
     7921 atacattaat aggtacgaaa aagcaacttt ttttgcgctt aaaaccagtc ataccaataa
     7981 cttaagggta actagcctcg ccggcaatag ttacccttat tatcaagata agaaagaaaa
     8041 ggatttttcg ctacgctcaa atcctttaaa aaaacacaaa agaccacatt ttttaatgtg
     8101 gtcttttatt cttcaactaa agcacccatt agttcaacaa acgaaaattg gataaagtgg
     8161 gatattttta aaatatatat ttatgttaca gtaatattga cttttaaaaa aggattgatt
     8221 ctaatgaaga aagcagacaa gtaagcctcc taaattcact ttagataaaa atttaggagg
     8281 catatcaaat gaaatttaat aaaattgatt tagacaattg gaagagaaaa gagatattta
     8341 atcattattt gaaccaacaa acgactttta gtataaccac agaaattgat attagtgttt
     8401 tataccgaaa cataaaacaa gaaggatata aattttaccc tgcatttatt ttcttagtga
     8461 caagggtgat aaactcaaat acagctttta gaactggtta caatagcgac ggagagttag
     8521 gttattggga taagttagag ccactttata caatttttga tggtgtatct aaaacattct
     8581 ctggtatttg gactcctgta aagaatgact tcaaagagtt ttatgattta tacctttctg
     8641 atgtagagaa atataatggt tcggggaaat tgtttcccaa aacacctata cctgaaaatg
     8701 ctttttctct ttctattatt ccatggactt catttactgg gtttaactta aatatcaata
     8761 ataatagtaa ttaccttcta cccattatta cagcaggaaa attcattaat aaaggtaatt
     8821 caatatattt accgctatct ttacaggtac atcattctgt ttgtgatggt tatcatgcag
     8881 gattgtttat gaactctatt caggaattgt cagataggcc taatgactgg cttttataat
     8941 atgagataat gccgactgta ctttttacag tcggttttct aatgtcacta acctgccccg
     9001 ttagttgaag aaggttttta tattacagct ccagatccat atccttcttt ttctgaaccg
     9061 acttctcctt tttcgcttct ttattccaat tgctttattg acgttgagcc tcggaaccgg
     9121 tacccaggaa acagctatga ccatgtaata cgactcacta tacggggata tcgtcggaat
     9181 tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt
     9241 tcttgccgcc aaggatctga tggcgcaggg gatcaagatc tgatcaagag acaggatgag
     9301 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     9361 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     9421 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     9481 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     9541 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     9601 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     9661 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     9721 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     9781 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     9841 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     9901 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     9961 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
    10021 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
    10081 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgagagct cgcttggact
    10141 cctgttgata gatccagtaa tgacctcaga actccatctg gatttgttca gaacgctcgg
    10201 ttgccgccgg gcgtttttta ttggtgagaa tccaagcact
//