LOCUS       Exported                7664 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  gRNA expression empty vector (yeast).
ACCESSION   .
VERSION     .
KEYWORDS    pRPR1_gRNA_handle_RPR1t
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7664)
  AUTHORS   Farzadfard F, Perli SD, Lu TK
  TITLE     Tunable and Multifunctional Eukaryotic Transcription Factors Based 
            on CRISPR/Cas.
  JOURNAL   ACS Synth Biol. 2013 Sep 11.
  PUBMED    23977949
REFERENCE   2  (bases 1 to 7664)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2013 Sep 11."
FEATURES             Location/Qualifiers
     source          1..7664
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     77..94
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     322..342
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        324..342
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        365..771
                     /gene="RPR1"
                     /label=RPR1 promoter
                     /note="promoter for the S. cerevisiae RNase P RNA gene"
     misc_RNA        779..854
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      867..929
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     primer_bind     complement(1318..1337)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1319..1337)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1347..1364)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1347..1363)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1356..1378)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1504..1959
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1591..1610)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1801..1822
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2260..2667
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2668..3762
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(4256..4275)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4375..4397
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4435..4453)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4494..5836
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    4826..4873
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        5863..5967
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5968..6828
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6186..6205)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6999..7587
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7488..7507
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"