LOCUS       Exported                4592 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector P1-EYFP-Triplex-28-gRNA2-28-pA, complete sequence.
ACCESSION   KJ796501
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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4592
                     /organism="Cloning vector P1-EYFP-Triplex-28-gRNA2-28-pA"
                     /mol_type="other DNA"
                     /db_xref="taxon:1520549"
     rep_origin      complement(99..687)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(858..1718)
                     /codon_start=1
                     /product="ampicillin resistance protein"
                     /label=ampicillin resistance protein
                     /protein_id="AIJ03100.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(1719..1823)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1850..2305
                     /direction=RIGHT
                     /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
                     /regulatory_class="promoter"
                     /note="Minimal Ade MLP"
     CDS             3037..3741
                     /codon_start=1
                     /product="Phialidium yellow fluorescent proteim"
                     /label=PhiYFP
                     /note="mammalian codon-optimized"
                     /translation="MSSGALLFHGKIPYVVEMEGDVDGHTFSIRGKGYGDASVGKVDAQ
                     FICTTGDVPVPWSTLVTTLTYGAQCFAKYGPELKDFYKSCMPDGYVQERTITFEGDGNF
                     KTRAEVTFENGSVYNRVKLNGQGFKKDGHVLGKNLEFNFTPHCLYIWGDQANHGLKSAF
                     KICHEIAGSKGDFIVADHTQMNTPIGGGPVHVPEYHHMSYHVKLSKDVTDHRDNMSLTE
                     TVRAVDCRKTYL"
     gene            3037..3738
                     /gene="EYFP"
                     /label=EYFP
     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_feature    3939..4042
                     /label=gRNA2
                     /note="gRNA2"
     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)"
     regulatory      4137..4358
                     /regulatory_class="polyA_signal_sequence"
                     /gene="SV40"
     gene            4137..4358
                     /gene="SV40"
                     /label=SV40
     polyA_signal    4146..4267
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"