LOCUS       62056_4135        9926 bp DNA     circular SYN 28-JUN-2019
DEFINITION  Cloning vector pBM062, complete sequence.
ACCESSION   MN019114
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9926)
  AUTHORS   Markus BM, Bell GW, Lorenzi HA, Lourido S.
  TITLE     Optimizing Systems for Cas9 Expression in Toxoplasma gondii
  JOURNAL   mSphere 4 (3), e00386-19 (2019)
  PUBMED    31243081
REFERENCE   2  (bases 1 to 9926)
  AUTHORS   Markus BM.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-JUN-2019) Whitehead Institute, 455 Main Street, 
            Cambridge, MA 02142, United States
REFERENCE   3  (bases 1 to 9926)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "mSphere"; 
            date: "2019"; volume: "4"; issue: "3"; pages: "e00386-19"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (04-JUN-2019) Whitehead Institute, 455 Main Street, Cambridge, MA 
            02142, United States"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..9926
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     5'UTR           651..1193
                     /label=TUB1-cat-SaG1
                     /note="TUB1-cat-SaG1"
     CDS             1200..1853
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     3'UTR           1857..2185
                     /label=SAG1
                     /note="SAG1"
     misc_feature    2197..2696
                     /label=U6 upstream region
                     /note="U6 upstream region"
     misc_feature    2697..2717
                     /label=sgRNA-1 protospacer
                     /note="sgRNA-1 protospacer"
     misc_RNA        2718..2793
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2794..2808
                     /label=PolIII terminator
                     /note="PolIII terminator"
     5'UTR           2821..3319
                     /label=TgTUB1
                     /note="TgTUB1"
     misc_feature    3324..3389
                     /note="3xFLAG; three tandem FLAG(R) epitope tags, followed
                     by an enterokinase cleavage site"
     misc_feature    3396..3416
                     /note="SV40 NLS; nuclear localization signal of SV40 large
                     T antigen"
     CDS             3441..7541
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     misc_feature    7542..7589
                     /note="nucleoplasmin NLS; bipartite nuclear localization
                     signal from nucleoplasmin"
     polyA_signal    7623..7830
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7839..7979
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      8054..8509
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        8791..8895
                     /label=AmpR promoter
     CDS             8896..9753
                     /label=AmpR
                     /note="beta-lactamase"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cgggcccccc
      661 ctcgaggtcg acggtatcga taagcttgat ggcgatgcat gtcccgcgtt cgtgaaattc
      721 tctgcatcag cggagtgatc aggaatcatc gtctcagcgg gatgacgttg cggagcaggc
      781 cggctcgcgg tgggcagtca gatgccgaag gcgtaactca ggacggcttg cgctcatcgc
      841 agaacagggg tggtgcctgc attgggtgcg gttggtgatc ctggttggac cggtggagat
      901 gcgcgcgcac gaaggggatg tgtcagaaac attttgtttg ttctctgtga acttttagat
      961 gtgttaaagg cggcgaatat tancagagag tcctccttgt tggattctct cttgaatttc
     1021 gccctttcct tctctttgcg agtctcgtag agaacaagca ctcgttcgcc gtccctgacg
     1081 acgcaacccg cgcagaagac atccaccaaa cggtgttaca caatcacctt gtgtgaagtt
     1141 cttgcggaaa actactcgtt ggcatttttt cttgaattcc ctttttcgac aaaatgcatg
     1201 agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt
     1261 ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta
     1321 cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca
     1381 ttcttgcccg cctgatgaat gctcatccgg aattccgtat ggcaatgaaa gacggtgagc
     1441 tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt
     1501 tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc
     1561 aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata
     1621 tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca
     1681 atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca
     1741 aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg
     1801 gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg gcttaattaa
     1861 tcaccgttgt gctcacttct caaatcgaca aaggaaacac acttcgtgca gcatgtgccc
     1921 cattataaag aaactgagtt gttccgttgt ggcttgcagg tgtcacatcc acaaaaaccg
     1981 gccgactcta aataggagtg tttcgcagca agcagcgaaa gtttatgact gggtccgaat
     2041 ctctgaacgg atgtgtggcg gacctggctg atgttgatcg ccgtcgacac acgcgccaca
     2101 tgggtcaata cacaagacag ctatcagttg ttttagtcga accggttaac acaattcttg
     2161 cccccccgag ggggatccac tagttctaga ggtaccgatg agacaaagtg cgcgagttga
     2221 aatcgtcgtg gggacgattt caccgcggcc acatgttgga gacactgagg gcacacggga
     2281 aacgcgaaag atttcaaatt aacgtaccca aacgcgaaag cttgcgcagc atacactcga
     2341 agcgaacatc ccgaaccatc gagaggcgga gagcgataag tctttcacgc tgcgaagtgt
     2401 tgcgacggct gcgccgctgc actgtgaatt gggcgccaat attgcatcct aggcctgacg
     2461 cgcctcctgc agaacgcgag acactgggat atgtagagcc aagggggaaa ccttcgaact
     2521 ctcgaatgtc ttctctgaca agaatcatat ttccatcagt tctgtcagat tttcaaatgg
     2581 cgacctgcag aggcctgctt cctccctgtg cgctcttcga aggggctttc tgtcgcgcag
     2641 ggtcacctcg tccccgaagg gggtgtttgc cttctggtaa atggggatgt caagttgaga
     2701 atgcagttta gcaccgtgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     2761 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ttctttttct ctagaggtac
     2821 catgcatgtc ccgcgttcgt gaaattctct gcatcagcgg agtgatcagg aatcatcgtc
     2881 tcagcgggat gacgttgcgg agcaggccgg ctcgcggtgg gcagtcagat gccgaaggcg
     2941 taactcagga cggcttgcgc tcatcgcaga acaggggtgg tgcctgcatt gggtgcggtt
     3001 ggtgatcctg gttggaccgg tggagatgcg cgcgcacgaa ggggatgtgt cagaaacatt
     3061 ttgtttgttc tctgtgaact tttagatgtg ttaaaggcgg cgaatattan cagagagtcc
     3121 tccttgttgg attctctctt gaatttcgcc ctttccttct ctttgcgagt ctcgtagaga
     3181 acaagcactc gttcgccgtc cctgacgacg caacccgcgc agaagacatc caccaaacgg
     3241 tgttacacaa tcaccttgtg tgaagttctt gcggaaaact actcgttggc attttttctt
     3301 gaattccctt tttcgacacc atggactata aggaccacga cggagactac aaggatcatg
     3361 atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg aaggtcggta
     3421 tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc ggcaccaact
     3481 ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc
     3541 tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca
     3601 gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac
     3661 ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg
     3721 acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc
     3781 ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca
     3841 tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct
     3901 atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga
     3961 accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc
     4021 tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca
     4081 gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga
     4141 atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca
     4201 acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc
     4261 tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga
     4321 acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg
     4381 cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc
     4441 tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga
     4501 gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt
     4561 tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca
     4621 gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc
     4681 acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg
     4741 acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc
     4801 tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc
     4861 cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga
     4921 tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt
     4981 acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga
     5041 gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga
     5101 ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct
     5161 tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc
     5221 acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca
     5281 ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac
     5341 ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga
     5401 gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt
     5461 ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc
     5521 agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg
     5581 gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga
     5641 agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca
     5701 agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga
     5761 agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga
     5821 tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact
     5881 acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg
     5941 actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca
     6001 aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg
     6061 tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga
     6121 gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg
     6181 gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc
     6241 tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag
     6301 tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag
     6361 tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa
     6421 ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg
     6481 tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca
     6541 agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg
     6601 gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg
     6661 ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg
     6721 tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga
     6781 acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg
     6841 acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca
     6901 agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg
     6961 agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga
     7021 tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg
     7081 cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact
     7141 tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga
     7201 aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg
     7261 agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca
     7321 acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc
     7381 tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga
     7441 ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc
     7501 tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg gcggccacga
     7561 aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcct agagctcgct gatcagcctc
     7621 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     7681 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     7741 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     7801 ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt
     7861 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     7921 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     7981 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     8041 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     8101 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     8161 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     8221 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     8281 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     8341 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat
     8401 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     8461 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     8521 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     8581 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     8641 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     8701 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     8761 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     8821 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     8881 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     8941 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     9001 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     9061 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     9121 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     9181 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     9241 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     9301 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     9361 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     9421 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     9481 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     9541 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     9601 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     9661 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     9721 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     9781 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     9841 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     9901 cccgtagaaa agatcaaagg atcttc
//
