LOCUS       62056_4130        9826 bp DNA     circular SYN 28-JUN-2019
DEFINITION  Cloning vector pBM019, complete sequence.
ACCESSION   MN019123
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9826)
  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 9826)
  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 9826)
  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..9826
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(4..861)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(862..966)
                     /label=AmpR promoter
     rep_origin      complement(992..1447)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     1589..1605
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1612..1630
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1656..1672
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1684..2183
                     /label=U6 upstream region
                     /note="U6 upstream region"
     misc_feature    2184..2204
                     /label=sgRNA-1 protospacer
                     /note="sgRNA-1 protospacer"
     misc_RNA        2205..2280
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2281..2295
                     /label=PolIII terminator
                     /note="PolIII terminator"
     5'UTR           2309..2806
                     /label=TgTUB1
                     /note="TgTUB1"
     misc_feature    2816..2881
                     /note="3xFLAG; three tandem FLAG(R) epitope tags, followed
                     by an enterokinase cleavage site"
     misc_feature    2888..2908
                     /note="SV40 NLS; nuclear localization signal of SV40 large
                     T antigen"
     CDS             2933..7033
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     misc_feature    7034..7081
                     /note="nucleoplasmin NLS; bipartite nuclear localization
                     signal from nucleoplasmin"
     CDS             7097..7153
                     /label=P2A
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
     CDS             7169..7822
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     CDS             7823..7849
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     3'UTR           7859..8601
                     /label=DHFR
                     /note="DHFR"
     promoter        complement(8627..8645)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8666..8682)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(8690..8706)
                     /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)."
     promoter        complement(8714..8744)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(8759..8780)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(9068..9656)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     1021 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     1081 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     1141 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     1201 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     1261 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     1321 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     1381 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     1441 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     1501 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     1561 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac
     1621 tcactatagg gcgaattggg taccgggccc cccctcgagg tcgacggtat cgatattaat
     1681 taagatgaga caaagtgcgc gagttgaaat cgtcgtgggg acgatttcac cgcggccaca
     1741 tgttggagac actgagggca cacgggaaac gcgaaagatt tcaaattaac gtacccaaac
     1801 gcgaaagctt gcgcagcata cactcgaagc gaacatcccg aaccatcgag aggcggagag
     1861 cgataagtct ttcacgctgc gaagtgttgc gacggctgcg ccgctgcact gtgaattggg
     1921 cgccaatatt gcatcctagg cctgacgcgc ctcctgcaga acgcgagaca ctgggatatg
     1981 tagagccaag ggggaaacct tcgaactctc gaatgtcttc tctgacaaga atcatatttc
     2041 catcagttct gtcagatttt caaatggcga cctgcagagg cctgcttcct ccctgtgcgc
     2101 tcttcgaagg ggctttctgt cgcgcagggt cacctcgtcc ccgaaggggg tgtttgcctt
     2161 ctggtaaatg gggatgtcaa gttgagaatg cagtttagca ccgtgtttta gagctagaaa
     2221 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     2281 tttttttttc tttttggcgc gccattaaca tgcatgtccc gcgttcgtga aattctctgc
     2341 atcagcggag tgatcaggaa tcatcgtctc agcgggatga cgttgcggag caggccggct
     2401 cgcggtgggc agtcagatgc cgaaggcgta actcaggacg gcttgcgctc atcgcagaac
     2461 aggggtggtg cctgcattgg gtgcggttgg tgatcctggt tggaccggtg gagatgcgcg
     2521 cgcacgaagg ggatgtgtca gaaacatttt gtttgttctc tgtgaacttt tagatgtgtt
     2581 aaaggcggcg aatattanca gagagtcctc cttgttggat tctctcttga atttcgccct
     2641 ttccttctct ttgcgagtct cgtagagaac aagcactcgt tcgccgtccc tgacgacgca
     2701 acccgcgcag aagacatcca ccaaacggtg ttacacaatc accttgtgtg aagttcttgc
     2761 ggaaaactac tcgttggcat tttttcttga attccctttt tcgacacaat tgatggacta
     2821 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa
     2881 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa
     2941 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     3001 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     3061 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     3121 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     3181 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     3241 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     3301 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     3361 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     3421 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     3481 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     3541 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     3601 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     3661 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     3721 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     3781 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     3841 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     3901 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     3961 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     4021 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     4081 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     4141 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     4201 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     4261 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     4321 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     4381 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     4441 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     4501 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     4561 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     4621 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     4681 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     4741 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     4801 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     4861 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     4921 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     4981 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     5041 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     5101 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     5161 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     5221 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     5281 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     5341 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     5401 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     5461 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     5521 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     5581 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     5641 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     5701 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     5761 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     5821 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     5881 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     5941 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     6001 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     6061 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     6121 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     6181 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     6241 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     6301 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     6361 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     6421 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     6481 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     6541 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     6601 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     6661 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     6721 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     6781 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     6841 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     6901 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     6961 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     7021 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     7081 gcatatggga agcggagcta ctaacttcag cctgctgaag caggctggag acgtggagga
     7141 gaaccctgga cctgctagcg gaatgcatga gaaaaaaatc actggatata ccaccgttga
     7201 tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac
     7261 ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa
     7321 gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga
     7381 attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta
     7441 caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga
     7501 tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc
     7561 ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag
     7621 tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac
     7681 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
     7741 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
     7801 cgatgagtgg cagggcgggg cttacccata cgatgttcct gactatgcgt aaggatccac
     7861 agaagctgcc cgtctctcgt tttcctctct tttcggaggg atcagggaga gtgcctcggg
     7921 tcggagagag ctgacgaggg ggtgccagag acccctgtgt cctttatcga agaaaaggga
     7981 tgactcttca tgtggcattt cacacagtct cacctcgcct tgttttcttt ttgtcaatca
     8041 gaacgaaagc gagttgcggg tgacgcagat gtgcgtgtat ccactcgnga atgcgttatc
     8101 gttctgtatg ccgctagagt gctggactgt tgctgtctgc ccacgacagc agacaacttt
     8161 ccttctatgc acttgcagga tggtgcagcg caaacgacgg agagaaagga gcaccctctc
     8221 agtttcccta cgatgtgctg tcagtttcga ctcttcaccg cgaacgattg gcgatacgtc
     8281 tctgttgact tgttaggctc cgaccacgaa gctcccttaa ctagataagc cgcgacacct
     8341 aagtgtacac catttgcaga tcgataatct gcgaccgctg aatccgtcca gatcagtaaa
     8401 accgcaccac ctaagtgtaa accttgttta ggtcgataaa atgctaccaa cccccaccca
     8461 caatcgagcc ttgagcgttt ctgcgcacgc gttggcctac gtgacttgct gatgcctgcc
     8521 tctggccatt catgccagtc agtgcgcata aaaatgtgga cacagtcggt tgacaagtgt
     8581 tctggcaggc tacagtgaca cgcggccgca gctccagctt ttgttccctt tagtgagggt
     8641 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     8701 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     8761 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     8821 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     8881 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     8941 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     9001 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     9061 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     9121 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     9181 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     9241 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     9301 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     9361 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     9421 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     9481 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     9541 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     9601 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     9661 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     9721 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     9781 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacag
//
