LOCUS       40924_2353        4592 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector P1-EYFP-Triplex-28-gRNA2-28-pA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4592)
  AUTHORS   Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK.
  TITLE     Multiplexed and programmable regulation of gene networks with an 
            integrated RNA and CRISPR/Cas toolkit in human cells
  JOURNAL   Mol. Cell 54 (4), 698-710 (2014)
  PUBMED    24837679
REFERENCE   2  (bases 1 to 4592)
  AUTHORS   Perli SD.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-MAY-2014) Electrical Engineering and Computer Science,
            Massachusetts Institute of Technology, 77, Massachusetts Avenue, 
            Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 4592)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4592)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol. Cell";
            date: "2014"; volume: "54"; issue: "4"; pages: "698-710"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (04-MAY-2014) Electrical Engineering and Computer Science, 
            Massachusetts Institute of Technology, 77, Massachusetts Avenue, 
            Cambridge, MA 02139, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4592
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(99..687)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(861..1718)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(1719..1823)
                     /label=AmpR promoter
     rep_origin      1850..2305
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     misc_feature    2496..2587
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from
                     the human alpha-2 globin gene"
     misc_feature    2601..2962
                     /label=8x gRNA1 binding sites
                     /note="8x gRNA1 binding sites"
     regulatory      2969..3030
                     /label=Minimal Ade MLP
                     /note="Minimal Ade MLP"
                     /regulatory_class="promoter"
     CDS             3037..3738
                     /label=PhiYFP
                     /note="Phialidium yellow fluorescent proteim"
     misc_feature    3751..3860
                     /label=MALAT1_3 triplex
                     /note="MALAT1_3 triplex"
     misc_feature    3911..3938
                     /label=Csy4 rpt
                     /note="Csy4 rpt"
     protein_bind    3916..3930
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease
                     (Haurwitz et al., 2010)"
     misc_RNA        3959..4034
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    4043..4070
                     /label=Csy4 rpt
                     /note="Csy4 rpt"
     protein_bind    4048..4062
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease
                     (Haurwitz et al., 2010)"
     polyA_signal    complement(4146..4267)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
       61 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
      121 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
      181 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
      241 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
      301 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
      361 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
      421 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
      481 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
      541 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
      601 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
      661 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
      721 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
      781 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
      841 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
      901 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
      961 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     1021 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     1081 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     1141 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     1201 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     1261 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     1321 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     1381 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     1441 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     1501 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     1561 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     1621 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     1681 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     1741 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     1801 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt
     1861 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     1921 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     1981 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2041 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2101 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2161 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     2221 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     2281 aacaaaatat taacgtttac aatttcccat tcgccattca ggctgcgcaa ctgttgggaa
     2341 gggcgatcgg tgcgggcctc ttcgctatta cgccagccca agctaccatg ataagtaagt
     2401 aatattaagg tacgggaggt acttggagcg gccgcaataa aaatctttat ctcattacat
     2461 ctgtgtgttg gttttttgtg tgaatcgata gtactaacat acgctctcca tcaaaacaaa
     2521 acgaaacaaa acaaactagc aaaataggct gtccccagtg caagtgcagg tgccagaaca
     2581 tttctctatc gatagctagc tgaaatatta atgtcgcgac tattaatatt ttcaggctag
     2641 ccatgcttcg ctacacgcga ctattaatat tttcaggcta gccatgcttc gctacacgcg
     2701 actattaata ttttcaagct agccatgctt cgctacacgc gactattaat attttcaggc
     2761 tagccatgct tcgctaacgc gactattaat attttcaggc tagtcatgct tcgctacacg
     2821 cgactattaa tattttcagg ctagccatgc ttcgctacac gcgactatta atattttcag
     2881 gctagccatg cttcgctaca cgcgactatt aatattttca ggctagccat gcttcgctac
     2941 acgcgactat taatattttc aggctagcgg ggggctataa aagggggtgg gggcgttcgt
     3001 cctgctatct agcgtcgcgt tgaccatggc gccaccatga gcagcggcgc cctgctgttc
     3061 cacggcaaga tcccctacgt ggtggagatg gagggcgatg tggatggcca caccttcagc
     3121 atccgcggta agggctacgg cgatgccagc gtgggcaagg tggatgccca gttcatctgc
     3181 accaccggcg atgtgcccgt gccctggagc accctggtga ccaccctgac ctacggcgcc
     3241 cagtgcttcg ccaagtacgg ccccgagctg aaggatttct acaagagctg catgcccgat
     3301 ggctacgtgc aggagcgcac catcaccttc gagggcgatg gcaatttcaa gacccgcgcc
     3361 gaggtgacct tcgagaatgg cagcgtgtac aatcgcgtga agctgaatgg ccagggcttc
     3421 aagaaggatg gccacgtgct gggcaagaat ctggagttca atttcacccc ccactgcctg
     3481 tacatctggg gcgatcaggc caatcacggc ctgaagagcg ccttcaagat ctgccacgag
     3541 atcgccggca gcaagggcga tttcatcgtg gccgatcaca cccagatgaa tacccccatc
     3601 ggcggcggcc ccgtgcacgt gcccgagtac caccacatga gctaccacgt gaagctgagc
     3661 aaggatgtga ccgatcaccg cgataatatg agcctgacgg agaccgtgcg cgccgtggat
     3721 tgccgcaaga cctacctgtg ataaaccggt gattcgtcag tagggttgta aaggtttttc
     3781 ttttcctgag aaaacaacct tttgttttct caggttttgc tttttggcct ttccctagct
     3841 ttaaaaaaaa aaaagcaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     3901 cggtctgcga gttcactgcc gtataggcag ctaagaaagt aagtcggagt actgtcctgt
     3961 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     4021 caccgagtcg gtgctttttt tcgttcactg ccgtataggc agctaagaaa caaacaggaa
     4081 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac ggccggccgc ttcgagcaga
     4141 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     4201 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     4261 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     4321 ggttttttaa agcaagtaaa acctctacaa atgtggtaaa atcgataagg atcgagtatg
     4381 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     4441 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     4501 ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4561 gagcggtatc agctcactca aaggcggtaa ta
//
