LOCUS       62056_4145        5279 bp DNA     circular SYN 28-JUN-2019
DEFINITION  Cloning vector pBM065, complete sequence.
ACCESSION   MN019112
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5279)
  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 5279)
  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 5279)
  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..5279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(763..1554)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     promoter        complement(1555..1659)
                     /label=AmpR promoter
     primer_bind     1779..1795
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      1921..2420
                     /label=U6 promoter
                     /note="U6 promoter"
                     /regulatory_class="promoter"
     misc_feature    2421..2517
                     /label=G_sgRNA-1 scrambled
                     /note="G_sgRNA-1 scrambled"
     misc_feature    2518..2532
                     /label=PolIII terminator
                     /note="PolIII terminator"
     regulatory      2574..3072
                     /label=TUB1 promoter
                     /note="TUB1 promoter"
                     /regulatory_class="promoter"
     misc_feature    3067..3075
                     /label=toxoplasma Kozak
                     /note="toxoplasma Kozak"
     misc_feature    3079..3084
                     /label=BamHI site
                     /note="BamHI site"
     CDS             3085..3792
                     /label=mRuby3
                     /note="monomeric red fluorescent protein derived from
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
     misc_feature    3808..3864
                     /note="eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides; P2A; 2A peptide from porcine teschovirus-1 
                     polyprotein"
     CDS             3880..4533
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     3'UTR           4537..4852
                     /label=SAG1
                     /note="SAG1"
     primer_bind     complement(4976..4992)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt cagaagaact cgtcaagaag
      781 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
      841 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
      901 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
      961 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     1021 catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     1081 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     1141 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     1201 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     1261 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     1321 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc
     1381 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     1441 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     1501 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatactctt
     1561 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     1621 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     1681 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     1741 gaggcccttt cgtctcgcgc gtttcggtga tgacggtggt aaaacgacgg ccagtcaagg
     1801 cgattaagtt gggtaacgcc agggttttcc cagtcacgcg aattgggtac gcggccgcgg
     1861 tctccaatcg cgtctgcatt cgctccggtt ttgcacagcc gttctgtctc acgaggtacc
     1921 gatgagacaa agtgcgcgag ttgaaatcgt cgtggggacg atttcaccgc ggccacatgt
     1981 tggagacact gagggcacac gggaaacgcg aaagatttca aattaacgta cccaaacgcg
     2041 aaagcttgcg cagcatacac tcgaagcgaa catcccgaac catcgagagg cggagagcga
     2101 taagtctttc acgctgcgaa gtgttgcgac ggctgcgccg ctgcactgtg aattgggcgc
     2161 caatattgca tcctaggcct gacgcgcctc ctgcagaacg cgagacactg ggatatgtag
     2221 agccaagggg gaaaccttcg aactctcgaa tgtcttctct gacaagaatc atatttccat
     2281 cagttctgtc agattttcaa atggcgacct gcagaggcct gcttcctccc tgtgcgctct
     2341 tcgaaggggc tttctgtcgc gcagggtcac ctcgtccccg aagggggtgt ttgccttctg
     2401 gtaaatgggg atgtcaagtt gaactaaata gctggagaaa tgtgctcccg gaagaggatg
     2461 acgtcatata ggctacatac gtgtggttga ccaatggtcc tcaataaata ttagtgattt
     2521 ttttttcttt ttggcgcgcc aaggtatcgg tcgctaggtt cacgccctgc aggatgcatg
     2581 tcccgcgttc gtgaaattct ctgcatcagc ggagtgatca ggaatcatcg tctcagcggg
     2641 atgacgttgc ggagcaggcc ggctcgcggt gggcagtcag atgccgaagg cgtaactcag
     2701 gacggcttgc gctcatcgca gaacaggggt ggtgcctgca ttgggtgcgg ttggtgatcc
     2761 tggttggacc ggtggagatg cgcgcgcacg aaggggatgt gtcagaaaca ttttgtttgt
     2821 tctctgtgaa cttttagatg tgttaaaggc ggcgaatatt agcagagagt cctccttgtt
     2881 ggattctctc ttgaatttcg ccctttcctt ctctttgcga gtctcgtaga gaacaagcac
     2941 tcgttcgccg tccctgacga cgcaacccgc gcagaagaca tccaccaaac ggtgttacac
     3001 aatcaccttg tgtgaagttc ttgcggaaaa ctactcgttg gcattttttc ttgaattccc
     3061 tttttcgaca aaatggcggg atccgtgtct aagggcgaag agctgatcaa ggaaaatatg
     3121 cgtatgaagg tggtcatgga aggttcggtc aacggccacc aattcaaatg cacaggtgaa
     3181 ggagaaggca gaccgtacga gggagtgcaa accatgagga tcaaagtcat cgagggagga
     3241 cccctgccat ttgcctttga cattcttgcc acgtcgttca tgtatggcag ccgtaccttt
     3301 atcaagtacc cggccgacat ccctgatttc tttaaacagt cctttcctga gggttttact
     3361 tgggaaagag ttacgagata cgaagatggt ggagtcgtca ccgtcacgca ggacaccagc
     3421 cttgaggatg gcgagctcgt ctacaacgtc aaggtcagag gggtaaactt tccctccaat
     3481 ggtcccgtga tgcagaagaa gaccaagggt tgggagccta atacagagat gatgtatcca
     3541 gcagatggtg gtctgagagg atacactgac atcgcactga aagttgatgg tggtggccat
     3601 ctgcactgca acttcgtgac aacttacagg tcaaaaaaga ccgtcgggaa catcaagatg
     3661 cccggtgtcc atgccgttga tcaccgcctg gaaaggatcg aggagagtga caatgaaacc
     3721 tacgtagtgc aaagagaagt ggcagttgcc aaatacagca accttggtgg tggcatggac
     3781 gagctgtaca agcatatggg aagcggagct actaacttca gcctgctgaa gcaggctgga
     3841 gacgtggagg agaaccctgg acctgctagc ggaatgcatg agaaaaaaat cactggatat
     3901 accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt
     3961 gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta
     4021 aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat
     4081 gctcatccgg aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt
     4141 cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa
     4201 taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt
     4261 gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat
     4321 ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc
     4381 cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg
     4441 attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta
     4501 caacagtact gcgatgagtg gcagggcggg gcttaagtga tcaccgttgt gctcacttct
     4561 caaatcgaca aaggaaacac acttcgtgca gcatgtgccc cattataaag aaactgagtt
     4621 gttccgttgt ggcttgcagg tgtcacatcc acaaaaaccg gccgactcta aataggagtg
     4681 tttcgcagca agcagcgaaa gtttatgact gggtccgaat ctctgaacgg atgtgtggcg
     4741 gacctggctg atgttgatcg ccgtcgacac acgcgccaca tgggtcaata cacaagacag
     4801 ctatcagttg ttttagtcga accggttaac acaattcttg cccccccgag ggactagttg
     4861 caggccacct gcgacagacg agccggcacc caccgacaaa cggcctgtga gacccaattg
     4921 tctggacggt cgacggtatc gataagcttg taacttgttt aatcatctca cagtggtcat
     4981 agctgtttcc tgattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5041 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5101 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5161 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5221 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgtt
//
